KR102021263B1 - PHARMACEUTICAL COMPOSITION Porphyra dentata Kjellman EXTRACT FOR SKIN REGENERATION. - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for skin regeneration containing a laver extract having enhanced single compound, dehydrated P-334, which is an active material contained in porphyra dentata kjellman, as an active ingredient. According to the present invention, single compounds, P-334 and DP-334, isolated from a laver promote the cell growth of skin keratinocyte (HACAT cell). The DP-334 which is a single compound isolated from the laver increases the expression of protein of epidermal growth factor receptor (EGFR), which is a receptor for sensing epidermal growth hormone, and mRNA, thereby confirming an effect of skin regeneration. Accordingly, the single compound, DP-334, isolated from the laver can be used in the pharmaceutical composition for skin regeneration having an effect on skin regeneration.

Description

PHARMACEUTICAL COMPOSITION Porphyra dentata Kjellman EXTRACT FOR SKIN REGENERATION.

The present invention relates to a pharmaceutical composition for skin regeneration comprising an active ingredient extracted and separated from itbide. More specifically, the present invention relates to a pharmaceutical composition for skin regeneration that can be safely used without toxicity and side effects using a compound isolated from seaweed, which is a natural raw material.

Skin is the largest tissue in the envelope system, which consists of a number of epithelial tissues that protect the muscles and organs in the body. The skin is located on the outermost side of the body to protect the body from pathogens when it encounters the external environment, and performs insulation, thermoregulation, sensory function, synthesis of vitamin D and protection of vitamin B folate.

The skin consists of the epidermis, which is the stratified squamous epithelium, the dermis, which is a tight connective tissue, and the subcutaneous fat layer, which is a loose connective tissue. The epidermal layer is the outermost part of the skin, creating a waterproof, protective membrane that covers the surface of the body and consists of a layered scale epithelial cell and the underlying layer. There is no blood vessel in the epidermis and there is a weak nerve associated with the central nervous system. The main types of cells that make the epidermis are keratinocytes, melanocytes, Langerhans cells and Merkel cells.

The dermis buffers the skin layer below the epidermis, which is made up of connective tissue, protecting the body from pressure and tension. The dermis is tightly connected to the epidermis through the basement membrane to support and feed the epidermis and contain multiple nerve endings that sense contact and heat. It interacts with the epidermis to help the skin regenerate, which is the component of cellular communication. The stratum corneum is the outermost component of the skin's epidermis and is made up of protein-rich keratinocytes. When the skin barrier is damaged, many keratin starts to occur. At this time, it is necessary to moisturize the skin with ingredients that can suppress the damage of the skin barrier.

Meanwhile, seaweed belongs to Phylum Rhodophyta, Class Bangiophyceae, Order Bangiales, and Family Bangiaceal, and is divided into Genus Porphyra and Genus Pyropia. In Korea, seven species of gimsok and 13 species of gimsok are reported to inhabit. Kim is a major producer and consumer of Japan and Korea. In 2012, the UPOV (International Union for the Protection of New Varieties of Plants) agreement expands the protection of varieties to all plants, including algae.

Japan is the main product of Porphyra yezoensis, China, the emerging producer of laver, is increasing the production of P.yezoensis, and Korea is P. tenera, Radiant laver, It body Produces P. dentata and P. seriata.

In particular, P. dentata Kjellman is a crude species that grows in the early stages of farming, and since no (neutral) spores are released, the farming is terminated after one or two harvesting and the production cannot be continued. As a result, bite production and price are unstable every year.

Its body of spinach is dark reddish, membranous, thin paper-like, with long dangular shape, and the lower part is blunt and the upper part is narrow. The cells on the surface are paired with the shape of the eyebrows and the shape of the pupil, but the two cells are not very close. The edges of the plant are uniquely bounded, with slender, bead-shaped cells. Sperm sac mainly divided into eight. Grow above the intertidal zone.

Therefore, while researching the efficacy of natural products such as itbim, there are few side effects on the skin and trying to develop a substance that helps to recover. However, there is a lack of relevant information on the beauty and bioactive function of itbimil. .

The present inventors conducted a study on skin regeneration of seaweed and found that the single compounds Porphyra-334 (P-334) and Dehydrated Porphyra-334 (DP-334) isolated from seaweed are effective substances showing major effects. An efficacy test was conducted.

Korean Laid-Open Patent Publication No. 10-2014-0097865 discloses excellent ability to inhibit tyrosinase activity, melanin synthesis, collagen synthesis, and MMP activity, keratinocytes (HaCaT), human fibroblasts (1064 SK), and skin cancer cells (B16F10). ) Whitening or wrinkles containing ginseng extract as an active ingredient, which shows little cytotoxicity, is non-irritating, very sensitive (grade I), and does not cause phototoxicity and photosensitization. Disclosed is a cosmetic composition for improvement. Korean Laid-Open Patent Publication No. 10-2016-0089911 includes the steps of: (a) first eluting an active ingredient of seaweed in distilled water; (b) separating the primary supernatant from the eluate and then eluting the residue again in distilled water; (c) separating the secondary supernatant from the secondary eluate; (d) mixing the first and second eluates and then filtering; And (e) sterilizing the filtrate and diluting the salinity to 0.7 to 1.2 psu. The present invention relates to a method for producing a taurine and alanine-containing laver extract and a cosmetic composition comprising the same. Korean Patent No. 10-1195577 discloses a cosmetic composition for alleviating skin inflammation, which comprises a mixed extract consisting of laver or radish larvae extract of red algae, mushroom mushroom (Sparassis crispa), agaricus mushroom (Agaricus blazeii), and lye (Polyporus umbellatus) Is disclosed. Korean Patent No. 10-1128591 discloses marine red algae extract mixture and cosmetics containing the same, which absorbs ultraviolet rays A, inhibits direct irradiation of ultraviolet rays to the skin, prevents oxidation by ultraviolet rays, and restores skin damage through cell regeneration. Disclosed is a composition. However, the above-mentioned prior art documents on the skin regeneration of the single body porphyra-334 (P-334) and Dehydrated Porphyra-334 (DP-334) to be presented in the present invention as a content of the simple extract and the sun protection Research is different from content.

In order to solve the problem of lack of relevant information on the beauty and physiological activity of seaweed, the task is to provide a pharmaceutical composition for skin regeneration that can be safely used without side effects using a single compound isolated from natural raw itbim gimyeo do.

The present invention provides a pharmaceutical composition for skin regeneration containing a hot water extract itbadi Seaweed (Porphyra. Dentata) as an active ingredient. In order to solve the above problems, there is provided a composition comprising a single compound Porphyra-334 (Porphyra-334) or a single compound Dehydrated P334 isolated from the it body turning as an active ingredient.

Itbody Spinning Extract The single compound P-334 (Porphyra-334) involves the change of activity to DP-334 (Dehydrated. Porphyra-334) through the extraction process. The composition of the present invention has the effect of promoting skin cell regeneration, promoting wound healing, the composition contains an amount of 0.001 ~ 2000 mg / kg weight with respect to dry weight, powders, granules, tablets, capsules, emulsions, It may be formulated as a syrup, transdermal, suppository or sterile injectable, band, ointment.

Single compounds, P-334 and DP-334, isolated from it-bodied spin, increased the growth of HACAT cells by increasing p- ERK protein expression associated with skin regeneration in in vitro experiments. 334 confirmed the skin regeneration effect by increasing the protein and mRNA expression of Epidermal Growth Factor Receptor (EGFR), a receptor that detects epidermal growth hormone. Therefore, it was confirmed that the single compound isolated from seaweed has an excellent effect as a pharmaceutical composition for skin regeneration.

1 is a diagram illustrating an extraction process of single-body turning compound (P334, DP334).
Figure 2 is a diagram showing the structure of the itbodied stone single compound (P334, DP334).
Figure 3 is a graph showing the cell growth change of keratinocytes for it body turning single compounds (P334, DP334).
Figure 4 is a diagram showing the change in the protein expression of keratinocytes for it body turning single compounds (P334, DP334).
5 is a diagram showing the change in the expression of mRNA of the keratinocytes for the itbodied stone single compound (P334, DP334).

Hereinafter, with reference to the accompanying drawings and the specific configuration related to the pharmaceutical composition for skin regeneration containing a single compound isolated from the laver of the present invention as an active ingredient will be described in detail.

1. Preparation of gum body single compound P-334, DP-334

Figure 1 shows the extraction process of the itbimdol single compound. It body rotation according to extraction temperature and sample. Single compound P-334, Dehydrated. The extraction process to obtain the content of P-334 is shown.

100 g of itbodied laver was washed with distilled water to remove salt, then 4,000 ml of distilled water was added, and heated and extracted at 100 ° C. for 3 hours in a reflux extractor. Filter using filter paper (whatman No. 41) and the filtrate was depressurized and concentrated. The concentrated hot water extract was freeze-dried at -50 ° C for 48 hours using a freeze dryer. In the above manner, 2g (2%) of single-body turning compound P334 was obtained.

Dehydrated-p334 was obtained by heating and extracting the obtained itbimim concentrate concentrated at 180 ℃ for 1 hour using an ion extraction equipment. Filter using filter paper (Advantec 110mm) and the filtrate was reduced and concentrated. The concentrated hot water extract was freeze-dried at -50 ° C for 48 hours using a freeze dryer. In this way, it is a single compound dehydrated. 100 mg (0.1% of total weight) of P-334 was obtained and used as a sample of the present invention experiment.

Figure 2 is a diagram showing the structure of the itbodied stone single compound (P334, DP334). P334, one of the components of it body spinning, was extracted by heating at 180 ° C through ion extraction equipment. As a result, the hydroxyl group (Hydroxyl Group, -OH group) of the structure of it body was separated and DP-334 was structurally changed. And increased activity.

2. Changes in Cell Growth of HACAT Cells against Itbodies Root Extracts

Cell growth change experiments of HACAT cells against IBDI extract were incubated in 96 well plates and stabilized for 24 hours after attachment to wells of 1 × 10 ⁶ cells / well of HACAT cells. Samples were treated with DMEM medium without fetal bovine serum (FBS) at different concentrations and incubated at 37 ° C and 5% CO ₂ for 24 hours. Cell growth rate (Ez-cytox) measurement 10ul each of the reagents were incubated for 4 hours at 37 ℃, 5% CO ₂ and the absorbance was measured at 450nm.

Figure 3 is a graph showing the cell growth change of the keratinocytes for the itbodies turning single compound (P334, DP334) Its body turning single compound P-334, Dehydrated. When the concentration of P-334 was treated at 1, 5 and 10 ug / ml, it was confirmed that the cell growth of the single-body compound was increased.

3. Changes in protein expression of keratinocytes against it body extract

The protein expression change experiments of the keratinocytes against the itbide extract was carried out as follows. The cells are uniformly distributed in a 60mm dish with 3.5 × 10 ⁵ cells and stabilized for 24 hours. Deplete and sample after 24 hours. After 24 hours of sample treatment, wash twice with PBS and dissolve in 1x lysis buffer. After lysis for 30 minutes at room temperature, centrifuge for 15 minutes at 12000 rpm 4 ℃.

After centrifugation, only the supernatant is collected and used at -20 ° C. For immunoblotting, 25 µg / lane of protein was electrophoresed on a 10% SDS-Polyacrylamide gel, and the membrane was reacted for 1 hour at room temperature with 5% Skim milk. The primary antibody was treated at a ratio of 1: 1000 in Tris Buffered saline and washed three times in TBST (Tris Buffered saline-Tween 20) the next day.The secondary antibody was then washed with 3% skim milk in anti-rabbit or anti-mouse antibody (2 Primary antibody) is treated at 1: 1000 ratio for 1 hour, followed by immunoblotting.

Figure 4 is a diagram showing the change in protein expression of the keratinocytes for the itbimdol single compound. EGF10ng / ml as a positive control group for skin keratinocytes, single-body turning compound P-334, Dehydrated. Treatment of P-334 with 1, 5, and 10 ug / ml resulted in an increase in protein expression of P-ERK, which is involved in cell growth, by EGF 10 ng / ml.

In addition, the protein expression of EGFR (Epidermal Growth Factor Receptor), a receptor that detects epidermal growth hormone, is altered in Ib-dibody single compound P-334. There was no, and increased by concentration in Dehydrated.P-334.

4. Confirmation of gene expression related to skin regeneration factor through RT-PCR

In order to confirm gene expression related to skin regeneration factor by RT-PCR, HACAT cells, which are keratinocytes, were dispensed in a 60 mm plate with a cell number of 2 × 10 ⁵ cells / ml and incubated at 37 ° C. and 5% CO ₂ for 24 hours. It was. After pretreatment with 10 ng / ml EGF, it was treated with single body dehydrated. Dehydrated. P-334 1, 5, 10 ug / ml. After 24 hours of sample treatment, cartilage cells were recovered by washing with PBS (phosphate buffered saline) using 0.05% Trypsin-EDTA and a centrifuge.

The recovered cells were isolated from RNA using RNeasy Mini Kit (Qiagen). After quantification using isolated RNA, cDNA was synthesized and amplified using each primer, DEPC water, and TaKaRa Ex Taq (TaKaRa). Table 1 shows the primers for the expression of mRNA of keratinocytes for the itbodied turning single compound (P334, DP334).

Primer for the expression of mRNA of keratinocytes against itbody turning single compounds (P334, DP334). EGFR sense: 5'-CTTGCAGCGATACAGCTCAG-3 '
antisense: 5-GGCTGTCGAATGTGCTGTTTG-3 ' Beta-actin sense: 5'-AGCGAGCATCCCCCAAAGTT-3 '
antisense: 5'-GGGCACGAAGGCTCATCATT-3 '

-EGFR primer: Expression of Epidermal Growth Factor Receptor (EGFR) and Autotaxin (ENPP2) in Two Phenotypically Different Neuroblastoma Cell Lines

Beta-actin primer: Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and their roles

In order to confirm the quantitative difference between the PCR products, 2% agarose gel was prepared with 1 × TAE buffer, DNA gel loading solution was mixed with PCR products corresponding to each primer per well, and electrophoresis was performed at 100 V. After staining with EtBr was confirmed whether the expression changes under UV. The housekeeping gene Beta-actin was used as internal control.

5 is a diagram showing the change in the expression of mRNA of the keratinocytes for the itbodied stone single compound (P334, DP334). The growth factor of EBB, Dehydrated.P-334 1, 5, and 10ug / ml, increased as the growth factor of EGF 10ng / ml was applied to skin regenerated cells (HACAT). The expression of EGFR of the single body dehydrated compound P-334 increased by concentration of 10 ng / ml EGF, a positive control.

The present invention increases the content of the active ingredient DP-334 involved in skin regeneration by extracting a pharmaceutical composition for skin regeneration containing single compounds isolated from seaweed as an active ingredient to regenerate the skin in HACAT Cells. There is industrial applicability as it can generate benefits and profits for products related to skin regeneration and related companies.

Claims (5)

It is a pharmaceutical composition for skin regeneration comprising a single compound Porphyra-334 (Porphyra-334) or a single compound Dehydrated P334 separated and extracted from the hot water extract of Porphyra. Dentata The pharmaceutical composition for skin regeneration according to claim 1, wherein the single compound P-334 (Porphyra-334) is structurally changed to DP-334 (Dehydrated. Porphyra-334) through an extraction process. The pharmaceutical composition for skin regeneration according to claim 1, wherein the composition has an effect of promoting skin cell regeneration and promoting wound healing. The composition according to claim 1, wherein the composition contains an amount of 0.001 to 2000 mg / kg by weight based on dry weight, powder, granule, tablet, capsule, emulsion, syrup, transdermal, suppository or sterile injectable, band , Pharmaceutical composition for skin regeneration characterized in that it is formulated as an ointment delete

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