WO2020175754A1 - Pharmaceutical composition and cosmetic composition for skin regeneration comprising active ingredient extracted and isolated from porphyra dentata - Google Patents
Pharmaceutical composition and cosmetic composition for skin regeneration comprising active ingredient extracted and isolated from porphyra dentata Download PDFInfo
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- WO2020175754A1 WO2020175754A1 PCT/KR2019/010853 KR2019010853W WO2020175754A1 WO 2020175754 A1 WO2020175754 A1 WO 2020175754A1 KR 2019010853 W KR2019010853 W KR 2019010853W WO 2020175754 A1 WO2020175754 A1 WO 2020175754A1
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
- A61K8/9717—Rhodophycota or Rhodophyta [red algae], e.g. Porphyra
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
Definitions
- the present invention relates to a pharmaceutical composition for skin regeneration and a functional cosmetic composition containing an active ingredient extracted and separated from the tooth body protrusion. More specifically, it relates to a pharmaceutical composition for skin regeneration and a functional cosmetic composition that can be safely used without toxicity and side effects by using a compound isolated from laver, a natural raw material.
- the skin is the largest tissue in the outer skin system consisting of a number of epithelial tissues that protect the muscles and organs in the body.
- the skin is located on the outermost part of the body and protects the body from pathogens when in contact with the external environment, and performs heat insulation, body temperature control, sensory functions, synthesis of vitamin D and protection of vitamin B folate.
- the skin consists of the epidermis, which is a middle-layer squamous epithelium, the dermis, which is a dense connective tissue, and the subcutaneous fat layer, which is a loose connective tissue.
- the epidermal layer is located on the outermost side of the skin and creates a waterproof and protective film covering the surface of the body, and consists of layered scaly epithelial cells and the underlying base layer.
- keratinocytes, melanocytes, Langerhans cells, and Merkel cells as the main types of cells that make up the epidermis.
- the dermis is a layer of skin underneath the epidermis made of connective tissue and acts as a buffer to protect the body from pressure and tension.
- the dermis is tightly connected to the epidermis through the basement membrane, supports the epidermis, supplies nutrients, and contains a plurality of nerve endings that sense contact and heat. It helps the skin regenerate through the interaction with the epidermis, and it is the cellular communication component that helps this interaction.
- 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, a lot of dead skin begins to occur, and at this time, it is necessary to moisturize the skin with ingredients that can suppress the damage to the skin barrier.
- Kim belongs to Phylum Rhodophyta, Class Bangiophyceae, Order Bangiales, and Family Bangiaceal, and can be divided into Genus Porphyra and Genus Pyropia. It has been reported that 7 species in seaweed and 13 species in stone seaweed in Korea. Korea and Japan are the major producers and consumers of Kim, and in Korea, the cultivar protection system will be expanded to all plants including seaweed in 2012 under the UPOV (International Union for the Protection of New Varieties of Plants) agreement.
- UPOV International Union for the Protection of New Varieties of Plants
- Japan's main product is Porphyra yezoensis
- the emerging laver producer China is increasing the production of P.yezoensis
- Korea is P. tenera, radiated laver, and itbody. It produces P. den insects and P. seriata.
- P. dentata Kjellman is an early-growing species that grows in the early stages of aquaculture, and due to the fact that short (neutral) spores are not released, the culture is terminated after harvesting once or twice. As a result, production and price of water laver are unstable every year.
- Itbodi protrusion plant is dark red, membranous, thin paper-like, long dengue shape, the lower part is blunt and the upper part is narrow.
- the cells seen from the surface form an eyebrow shape and a pupil shape, but the two cells are not very close.
- the edge of the plant is uniquely bounded, and the slim bead-shaped cells are sloppy.
- Sperm sac is mainly divided into eight. It grows in the upper intertidal zone.
- the present inventors conducted a study on the skin regeneration of laver, and found that the single compounds Porphyra-334 (P-334) and Dehydrated Porphyra-334 (DP-334) isolated from laver are effective substances showing major effects. The efficacy test was conducted on.
- a single compound isolated from the natural raw material, itbody provides a pharmaceutical composition for skin regeneration and a functional cosmetic composition that can be safely used without side effects. Make it your task.
- the invention itbadi Seaweed (Porphyra. Dentata) provides an effective pharmaceutical composition for skin regeneration and functional cosmetic composition containing a hot water extract or extract as an active ingredient.
- it provides an extract containing a single compound isolated from the tooth body protrusion as an active ingredient. It provides a composition comprising a single compound Porphyra-334 or a single compound Dehydrated P334 isolated from the protrusion of the tooth body as an active ingredient.
- Itbody protrusion extract contains a single compound Porphyra-334 or a single compound Dehydrated P334.
- Itbody extract single compound P-334 (Porphyra-334) includes the change and increase in activity to DP-334 (Dehydrated.Porphyra-334) through the extraction process.
- the composition of the present invention has an effect of promoting skin cell regeneration and promoting wound healing, and the composition contains an amount of 0.001 to 2000 mg/kg based on dry weight, and includes powders, granules, tablets, capsules, emulsions, It may be formulated as a syrup, transdermal, suppository or sterile injectable, band, or ointment.
- the single compounds P-334 and DP-334 isolated from the itbody protrusion increased the growth of skin keratinocytes (HACAT cells) by increasing the expression of p -ERK protein related to skin regeneration in an in vitro experiment, and DP- 334 was confirmed the skin regeneration effect by increasing protein and mRNA expression of EGFR (Epidermal Growth Factor Receptor), a receptor that detects epithelial growth hormone. Therefore, it was confirmed that the single compound isolated from laver exhibited excellent effects as a pharmaceutical composition for skin regeneration.
- EGFR Epimal Growth Factor Receptor
- FIG. 1 is a diagram showing the extraction process of a single compound (P334, DP334) of itbody protrusion.
- FIG. 2 is a diagram showing the structure of a single compound (P334, DP334).
- FIG. 3 is a graph showing changes in cell growth of skin keratinocytes for a single compound of itbody protrusion (P334, DP334).
- FIG. 4 is a diagram showing changes in protein expression of skin keratinocytes for a single compound (P334, DP334).
- Fig. 5 is a diagram showing changes in the expression of mRNA in keratinocytes for the single compound of itbody protrusion (P334, DP334).
- Dehydrated-p334 was extracted by heating at 180° C. for 1 hour using an ion extraction device to the obtained Itbody protrusion concentrate. Filtered 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. Dehydrated the single compound of the tooth body by the above method. 100 mg (0.1% of total weight) of P-334 was obtained and used as a sample of the experiment of the present invention.
- FIG. 2 is a diagram showing the structure of a single compound (P334, DP334).
- P334, DP334 a single compound.
- the hydroxyl group (Hydroxyl Group, -OH group) of the P334 structure of the monolithic product of the Itbody protrusion is removed, and the structural change to DP-334 And increased activity.
- HACAT cells skin keratinocytes
- FBS fetal bovine serum
- Figure 3 is a graph showing the change in the cell growth of skin keratinocytes with respect to a single compound (P334, DP334) of the tooth body protrusion, a single compound of the tooth body protrusion P-334, Dehydrated. As a result of treating the concentration of P-334 at 1, 5, and 10 ug/ml, it was confirmed that the cell growth of the single compound of the tooth body was increased.
- the experiment on the protein expression change of skin keratinocytes for the extract of ItBody Dolkim was carried out as follows.
- the cells are evenly distributed in a 60mm dish into 3.5 ⁇ 10 5 cells and then stabilized for 24 hours. Depletion and sample treatment after 24 hours. After 24 hours of sample treatment, wash twice with PBS and dissolve in 1x lysis buffer. After lysing the cells for 30 minutes at room temperature, centrifuge for 15 minutes at 12000 rpm at 4°C.
- FIG. 4 is a diagram showing the change in protein expression of keratinocytes for a single compound of itbody protrusion.
- EGF10ng/ml As a positive control for skin keratinocytes, EGF10ng/ml, itbody protrusion single compound P-334, Dehydrated.
- P-334 As a result of treatment with 1, 5, and 10 ug/ml of P-334, the protein expression of P-ERK involved in cell growth increased by concentration as much as EGF10 ng/ml, which is a positive control, in the Itbody protrusion single compound.
- cutaneous keratinocytes were dispensed into a 60mm plate at a cell number of 2 ⁇ 10 5 cells/ml, and cultured for 24 hours at 37°C and 5% CO 2 I did.
- EGF EGF
- it was treated with 1, 5, and 10 ug/ml of the single compound Dehydrated.P-334.
- chondrocytes were recovered using 0.05% Trypsin-EDTA and a centrifuge.
- 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
- a 2% agarose gel was made with 1 ⁇ TAE buffer, and a DNA gel loading solution was mixed and loaded with the PCR product corresponding to each primer per well, followed by electrophoresis at 100 V. After staining with EtBr, it was confirmed whether the expression was changed under UV. At this time, Beta-actin, a housekeeping gene, was used as an internal control.
- Fig. 5 is a diagram showing changes in the expression of mRNA in keratinocytes for the single compound of itbody protrusion (P334, DP334).
- the growth factor EGF 10ng/ml was treated in skin regeneration cells (HACAT)
- the itbody protrusion compound Dehydrated.P-334 1, 5 10ug/ml showed a tendency to increase by concentration.
- the present invention extracts a pharmaceutical composition for skin regeneration and a functional cosmetic composition containing a single compound isolated from laver as an active ingredient, thereby increasing the content of DP-334, an active ingredient involved in skin regeneration, so that skin keratinocytes (HACAT Cell) It has industrial applicability as it can produce skin regeneration effects and profits for products related to skin regeneration and related businesses.
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Abstract
The present invention relates to a pharmaceutical composition and functional cosmetic composition for skin regeneration comprising, as an active ingredient, a Porphyra dentata extract in which the single compound dehydrated P-334, which is an active ingredient contained in Porphyra dentata, is enhanced. Single compounds P-334 and DP-334 isolated from the seaweed according to the present invention promoted the growth of keratinocytes (HACAT cells). By confirming that DP-334, which is a single compound isolated from the seaweed, increases the protein and mRNA expression of the epidermal growth factor receptor (EGFR), which is a receptor for sensing epidermal growth hormones, thereby having a skin regeneration effect, the single compound DP-334 isolated from the seaweed according to the present invention can be used in a pharmaceutical composition and functional cosmetic compound for skin regeneration, which are effective in skin regeneration.
Description
본 발명은 잇바디 돌김에서 추출 분리한 유효성분을 함유하는 피부재생용 약학적 조성물 및 기능성 화장료 조성물에 관한 것이다. 보다 구체적으로는 천연원료인 김에서 분리한 화합물을 이용하여 독성 및 부작용 없이 안전하게 사용될 수 있는 피부재생용 약학적 조성물 및 기능성 화장료 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for skin regeneration and a functional cosmetic composition containing an active ingredient extracted and separated from the tooth body protrusion. More specifically, it relates to a pharmaceutical composition for skin regeneration and a functional cosmetic composition that can be safely used without toxicity and side effects by using a compound isolated from laver, a natural raw material.
피부는 체내의 근육들과 기관을 보호하는 다수의 상피 조직으로 이루어진 외피체계에서 가장 큰 조직이다. 피부는 몸의 가장 바깥쪽에 위치하여 외부환경을 접할 때에 병원균으로부터 신체를 보호하고, 단열과 체온조절기능, 감각기능, 비타민D의 합성과 비타민 B 엽산염의 보호 기능을 수행한다.The skin is the largest tissue in the outer skin system consisting of a number of epithelial tissues that protect the muscles and organs in the body. The skin is located on the outermost part of the body and protects the body from pathogens when in contact with the external environment, and performs heat insulation, body temperature control, sensory functions, synthesis of vitamin D and protection of vitamin B folate.
피부는 중층 편평 상피인 표피와 촘촘한 결합 조직인 진피(眞皮), 느슨한 결합조직인 피하지방층으로 이루어진다. 표피층은 피부의 가장 바깥쪽에 위치하고 신체의 표면을 덮는 방수, 보호 막을 만들며 층상의 비늘 상피세포와 그 밑의 기저층으로 이루어진다. 표피에는 혈관이 없고, 중추신경계와 관련되어 미약하게나 신경이 존재한다. 표피를 만드는 주된 세포들의 종류로 각질형성세포, 멜라닌세포, 랑게르한스세포, 메르켈 세포가 존재한다. The skin consists of the epidermis, which is a middle-layer squamous epithelium, the dermis, which is a dense connective tissue, and the subcutaneous fat layer, which is a loose connective tissue. The epidermal layer is located on the outermost side of the skin and creates a waterproof and protective film covering the surface of the body, and consists of layered scaly epithelial cells and the underlying base layer. There are no blood vessels in the epidermis, and nerves are weakly related to the central nervous system. There are keratinocytes, melanocytes, Langerhans cells, and Merkel cells as the main types of cells that make up the epidermis.
진피(dermis)는 연결조직으로 이루어진 표피 밑의 피부 층으로 완충작용을 하여 신체를 압력과 장력으로부터 보호한다. 진피는 기저막을 통해 표피와 단단히 연결되어 표피를 지지하고 영양분을 공급하며, 접촉과 열을 감지하는 복수의 신경 말단을 담고 있다. 표피와의 상호작용을 통해 피부의 재생을 돕는데, 이 상호 작용을 도와주는 것이 세포 대화 성분이다. 각질층은 피부 표피의 가장 바깥 부위에 있는 구성요소로 단백질이 풍부한 각질세포들로 이루어져 있다. 주로 피부 장벽이 손상된 경우 각질이 많이 생기기 시작하며, 이때 피부 장벽의 손상을 억제해줄 수 있는 성분들로 피부를 보습해줄 필요가 있다. The dermis is a layer of skin underneath the epidermis made of connective tissue and acts as a buffer to protect the body from pressure and tension. The dermis is tightly connected to the epidermis through the basement membrane, supports the epidermis, supplies nutrients, and contains a plurality of nerve endings that sense contact and heat. It helps the skin regenerate through the interaction with the epidermis, and it is the cellular communication component that helps this interaction. 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, a lot of dead skin begins to occur, and at this time, it is necessary to moisturize the skin with ingredients that can suppress the damage to the skin barrier.
한편, 김은 홍조식물문(Phylum Rhodophyta), 김파래홍조강(Class Bangiophyceae), 김파래목 (Order Bangiales), 김파래과(Family Bangiaceal)에 속하며, 김속(Genus Porphyra)과 돌김속(Genus Pyropia)로 나눌 수 있으며, 우리나라에는 김속에 7종, 돌김속에 13종이 서식하는 것으로 보고되어져 있다. 김은 한국과 일본이 주요 생산 및 소비국이며, 한국에서는 UPOV (International Union for the Protectionof New Varieties of Plants) 협약에 의해 2012년에 해조류를 비롯한 모든 식물로 품종보호제도가 확대된다. Meanwhile, Kim belongs to Phylum Rhodophyta, Class Bangiophyceae, Order Bangiales, and Family Bangiaceal, and can be divided into Genus Porphyra and Genus Pyropia. It has been reported that 7 species in seaweed and 13 species in stone seaweed in Korea. Korea and Japan are the major producers and consumers of Kim, and in Korea, the cultivar protection system will be expanded to all plants including seaweed in 2012 under the UPOV (International Union for the Protection of New Varieties of Plants) agreement.
일본은 방사무늬김(Porphyra yezoensis)을 주생산품으로 하며, 신흥 김 생산국인 중국은 방사무늬김(P.yezoensis)의 생산을 증대시키고 있으며, 한국은 참김(P. tenera), 방사무늬김, 잇바디돌김(P. dentata) 및 모무늬돌김(P. seriata)을 생산하고 있다.Japan's main product is Porphyra yezoensis, the emerging laver producer China is increasing the production of P.yezoensis, and Korea is P. tenera, radiated laver, and itbody. It produces P. dentata and P. seriata.
특히, 잇바디돌김(P. dentata Kjellman)은 조생종으로서 양식초기에 성장하며, 단(중성) 포자가 방출되지 않아 1∼2회 채취 후 양식이 종료되어 지속적인 생산이 이루어지지 못해 생산시기 및 생산량의 편중으로 해마다 물김 생산과 가격이 불안정하다.In particular, P. dentata Kjellman is an early-growing species that grows in the early stages of aquaculture, and due to the fact that short (neutral) spores are not released, the culture is terminated after harvesting once or twice. As a result, production and price of water laver are unstable every year.
잇바디 돌김 식물체는 검붉은색이며 막질이고 얇은 종이와 같으며 기다란 댕기모양으로 아랫부분은 뭉툭하고 윗부분은 좁아진다. 표면에서 보는 세포는 눈썹 모양인 것과 눈동자 모양인 것이 짝을 이루지만 그 두 세포 사이는 그다지 가깝지 않다. 식물체의 가장자리는 독특하게 경계를 이루며 갸름한 구슬모양의 세포들이 엉성하게 있다. 정자낭은 주로 8개로 나누어진다. 조간대 상부에 자란다. Itbodi protrusion plant is dark red, membranous, thin paper-like, long dengue shape, the lower part is blunt and the upper part is narrow. The cells seen from the surface form an eyebrow shape and a pupil shape, but the two cells are not very close. The edge of the plant is uniquely bounded, and the slim bead-shaped cells are sloppy. Sperm sac is mainly divided into eight. It grows in the upper intertidal zone.
따라서 잇바디 돌김과 같은 천연물의 효능을 연구하여 피부에 부작용이 거의 없고 회복에 도움이 되는 물질을 개발하기 위해 노력하고 있으나, 아직까지는 잇바디 돌김에 대한 미용 및 생리활성 기능성에 대한 관련정보가 부족한 실정이다.Therefore, by researching the efficacy of natural products such as the tooth body stone, we are striving to develop a substance that has few side effects on the skin and helps recovery, but there is still a lack of relevant information on the beauty and physiological activity of the tooth body stone. .
본 발명자는 김의 피부재생에 대한 연구를 수행하여 김에서 분리한 단일화합물 Porphyra-334 (P-334), Dehydrated Porphyra-334 (DP-334)가 주요효력을 나타내는 유효물질임을 찾게 되었으며, 피부재생에 대한 효력 시험을 진행하였다. The present inventors conducted a study on the skin regeneration of laver, and found that the single compounds Porphyra-334 (P-334) and Dehydrated Porphyra-334 (DP-334) isolated from laver are effective substances showing major effects. The efficacy test was conducted on.
김에 대한 미용 및 생리활성 기능성에 대한 관련정보가 부족한 문제점을 해결하기 위해서, 천연원료인 잇바디 돌김에서 분리한 단일 화합물을 이용하여 부작용 없이 안전하게 사용될 수 있는 피부재생용 약학적 조성물 및 기능성 화장료 조성물을 제공하는 것을 과제로 한다.In order to solve the problem of lack of relevant information on the beauty and physiological activity of seaweed, a single compound isolated from the natural raw material, itbody, provides a pharmaceutical composition for skin regeneration and a functional cosmetic composition that can be safely used without side effects. Make it your task.
본 발명은 잇바디 돌김(Porphyra. dentata) 열수 추출물 또는 추출물을 유효성분으로 함유하는 유효 피부재생용 약학적 조성물 및 기능성 화장료 조성물을 제공한다. 상기 과제를 해결하기 위하여, 잇바디 돌김에서 분리한 단일 화합물을 유효성분으로 함유하는 추출물을 제공한다. 잇바디 돌김에서 분리한 단일 화합물 포르피라-334(Porphyra-334) 또는 단일 화합물 Dehydrated P334를 유효성분으로 포함하는 조성물을 제공한다.The invention itbadi Seaweed (Porphyra. Dentata) provides an effective pharmaceutical composition for skin regeneration and functional cosmetic composition containing a hot water extract or extract as an active ingredient. In order to solve the above problems, it provides an extract containing a single compound isolated from the tooth body protrusion as an active ingredient. It provides a composition comprising a single compound Porphyra-334 or a single compound Dehydrated P334 isolated from the protrusion of the tooth body as an active ingredient.
잇바디 돌김 추출물은 단일 화합물 포르피라-334(Porphyra-334) 또는 단일 화합물 Dehydrated P334를 포함한다. 잇바디 돌김 추출 단일 화합물 P-334(Porphyra-334)는 추출과정을 통하여 DP-334 (Dehydrated.Porphyra-334)로 활성이 변화 및 증가되는 것을 포함한다. 본원발명의 상기 조성물은 피부 세포 재생 촉진, 상처 치유 촉진의 효능을 가지며, 상기 조성물은 건조 중량에 대하여 0.001~2000 mg/kg 중량의 양을 함유하며, 산제, 과립제, 정제, 캡슐제, 에멀젼, 시럽, 경피제, 좌제 또는 멸균용 주사용, 밴드, 연고로 제형화될 수 있다.Itbody protrusion extract contains a single compound Porphyra-334 or a single compound Dehydrated P334. Itbody extract single compound P-334 (Porphyra-334) includes the change and increase in activity to DP-334 (Dehydrated.Porphyra-334) through the extraction process. The composition of the present invention has an effect of promoting skin cell regeneration and promoting wound healing, and the composition contains an amount of 0.001 to 2000 mg/kg based on dry weight, and includes powders, granules, tablets, capsules, emulsions, It may be formulated as a syrup, transdermal, suppository or sterile injectable, band, or ointment.
잇바디 돌김에서 분리한 단일 화합물 P-334, DP-334는 in vitro 실험에서 피부재생과 관련된 p-ERK 단백질 발현을 증가시킴으로써 피부각질세포 (HACAT cell)의 성장을 증가시켰으며, 단일 화합물인 DP-334는 상피세포 성장 호르몬을 감지하는 수용체인 EGFR (Epidermal Growth Factor Receptor, 상피세포 성장 인자 수용체) 의 단백질 및 mRNA 발현을 증가시킴으로써 피부재생 효과를 확인하였다. 따라서 김에서 분리한 단일 화합물은 피부 재생용 약학적 조성물로서 우수한 효과를 나타내는 것으로 확인하였다. The single compounds P-334 and DP-334 isolated from the itbody protrusion increased the growth of skin keratinocytes (HACAT cells) by increasing the expression of p -ERK protein related to skin regeneration in an in vitro experiment, and DP- 334 was confirmed the skin regeneration effect by increasing protein and mRNA expression of EGFR (Epidermal Growth Factor Receptor), a receptor that detects epithelial growth hormone. Therefore, it was confirmed that the single compound isolated from laver exhibited excellent effects as a pharmaceutical composition for skin regeneration.
도 1은 잇바디 돌김 단일 화합물 (P334, DP334)의 추출과정을 나타낸 그림이다.FIG. 1 is a diagram showing the extraction process of a single compound (P334, DP334) of itbody protrusion.
도 2는 잇바디 돌김 단일 화합물 (P334, DP334)의 구조를 나타낸 그림이다.FIG. 2 is a diagram showing the structure of a single compound (P334, DP334).
도 3는 잇바디 돌김 단일 화합물(P334, DP334)에 대한 피부각질세포의 세포생장 변화를 나타낸 그래프이다.3 is a graph showing changes in cell growth of skin keratinocytes for a single compound of itbody protrusion (P334, DP334).
도 4은 잇바디 돌김 단일 화합물(P334, DP334)에 대한 피부각질세포의 단백질 발현의 변화를 나타낸 그림이다.FIG. 4 is a diagram showing changes in protein expression of skin keratinocytes for a single compound (P334, DP334).
도 5은 잇바디 돌김 단일 화합물(P334, DP334)에 대한 피부각질세포의 mRNA의 발현의 변화를 나타낸 그림이다.Fig. 5 is a diagram showing changes in the expression of mRNA in keratinocytes for the single compound of itbody protrusion (P334, DP334).
이하, 본 발명의 김에서 분리한 단일 화합물을 유효성분으로 함유하는 피부재생용 약학적 조성물 및 기능성 화장료 조성물과 관련한 구체적인 구성과 첨부한 도면을 참조하여 상세히 설명하면 다음과 같다.Hereinafter, with reference to the specific configuration of the pharmaceutical composition for skin regeneration and the functional cosmetic composition containing a single compound isolated from laver of the present invention as an active ingredient and the accompanying drawings, it will be described in detail as follows.
1. 잇바디 돌김 단일 화합물 P-334, DP-334 의 제조 1. Preparation of single compound P-334, DP-334
도 1은 잇바디 돌김 단일 화합물의 추출과정을 나타낸다. 추출 온도 및 시료에 따른 잇바디 돌김 단일 화합물 P-334, Dehydrated. P-334의 함량을 얻는 추출과정을 나타낸다. 1 shows the extraction process of a single compound of the itbody protrusion. It body protrusion single compound P-334, Dehydrated according to extraction temperature and sample. It shows the extraction process to obtain the content of P-334.
잇바디 돌김 100g을 염분을 제거하기 위하여 증류수로 수세한 다음, 증류수 4,000ml를 첨가하고, 환류 추출기에서 3시간 동안 100℃로 가열, 추출하였다. 여과지(whatman No.41)을 이용하여 여과하고 여액을 감압 및 농축하였다. 농축된 열수추출물을 동결건조기(freeze dryer)를 이용하여 -50℃에서 48시간 동안 동결 건조시켰다. 이상의 방법으로 잇바디 돌김 단일화합물 P334, 2g(2%)를 수득하였다. 100 g of Itbody protrusions were washed with distilled water to remove salt, and then 4,000 ml of distilled water was added, followed by heating and extraction at 100° C. for 3 hours in a reflux extractor. Filtered using filter paper (whatman No. 41), and the filtrate was reduced pressure and concentrated. The concentrated hot water extract was freeze-dried at -50°C for 48 hours using a freeze dryer. Itbody protrusions single compound P334, 2g (2%) was obtained by the above method.
Dehydrated-p334는 수득한 잇바디 돌김 농축액을 이온추출장비를 사용하여 1시간동안 180℃로 가열, 추출하였다. 여과지(Advantec 110mm)을 이용하여 여과하고 여액을 감압 및 농축하였다. 농축된 열수추출물을 동결건조기(freeze dryer)를 이용하여 -50℃에서 48시간 동안 동결 건조시켰다. 이상의 방법으로 잇바디 돌김 단일 화합물 Dehydrated. P-334 100mg(총 중량 대비 0.1%)를 수득하여 본 발명 실험의 시료로 사용하였다. Dehydrated-p334 was extracted by heating at 180° C. for 1 hour using an ion extraction device to the obtained Itbody protrusion concentrate. Filtered 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. Dehydrated the single compound of the tooth body by the above method. 100 mg (0.1% of total weight) of P-334 was obtained and used as a sample of the experiment of the present invention.
도 2는 잇바디 돌김 단일 화합물 (P334, DP334)의 구조를 나타낸 그림이다. 잇바디 돌김 성분중의 하나인 P334를 이온추출장비를 통하여 180℃에서 가열 추출한 결과, 잇바디 돌김 단일화화물의 P334 구조의 수산기 (Hydroxyl Group, -OH기)가 떨어져 나가게 되며, DP-334로 구조적인 변화 및 활성을 증가시켰다. FIG. 2 is a diagram showing the structure of a single compound (P334, DP334). As a result of heating and extracting P334, one of the components of the itbody protrusion, at 180℃ through the ion extraction equipment, the hydroxyl group (Hydroxyl Group, -OH group) of the P334 structure of the monolithic product of the Itbody protrusion is removed, and the structural change to DP-334 And increased activity.
2. 잇바디 돌김 추출물에 대한 피부각질세포 (HACAT cell) 의 세포생장 변화2. Changes in cell growth of skin keratinocytes (HACAT cells) in response to the extract of Itbody Dolkim
잇바디 돌김 추출물에 대한 피부각질세포 (HACAT cell) 의 세포생장 변화실험은 96 well plate에 1X106cells/well의 피부각질세포(HACAT cell)를 배양하여 well에 부착시킨 후 24시간 안정화시킨다. 소태아 혈청 (fetal bovine serum; FBS) 을 뺀 DMEM 배지와 함께 샘플을 농도 별로 처리하여 37℃, 5% CO2에서 24시간 배양한다. 세포생장률 (Ez-cytox) 측정 시약을 10ul씩 넣고 37℃, 5% CO2에서 4시간 동안 배양후 450㎚에서 흡광도를 측정하였다. In the experiment for cell growth change of skin keratinocytes (HACAT cells) for the extract of ItBody Dolkim, 1X10 6 cells/well of skin keratinocytes (HACAT cells) were cultured in a 96 well plate, attached to the well, and then stabilized for 24 hours. Samples were treated for each concentration with DMEM medium minus fetal bovine serum (FBS), and cultured at 37°C and 5% CO 2 for 24 hours. Cell growth rate (Ez-cytox) measuring reagents were added at a time of 10ul and incubated for 4 hours at 37°C and 5% CO 2 , and absorbance was measured at 450 nm.
도 3은 잇바디 돌김 단일 화합물(P334, DP334)에 대한 피부각질세포의 세포생장 변화를 나타낸 그래프로서 잇바디 돌김 단일화합물 P-334, Dehydrated. P-334 의 농도를 1, 5, 10ug/ml 로 처리한 결과, 잇바디 돌김 단일화합물의 세포생장이 증가하는 것을 확인할 수 있었다. Figure 3 is a graph showing the change in the cell growth of skin keratinocytes with respect to a single compound (P334, DP334) of the tooth body protrusion, a single compound of the tooth body protrusion P-334, Dehydrated. As a result of treating the concentration of P-334 at 1, 5, and 10 ug/ml, it was confirmed that the cell growth of the single compound of the tooth body was increased.
3. 잇바디 돌김 추출물에 대한 피부각질세포의 단백질 발현 변화3. Changes in protein expression of skin keratinocytes in the extract of Itbody Dolkim
잇바디 돌김 추출물에 대한 피부각질세포의 단백질 발현 변화실험은 다음과 같이 진행하였다. 세포를 60mm dish 에 3.5×105 세포로 균일하게 분포시킨 후 24시간 안정화 시킨다. 24시간 후 고갈 및 시료를 처리한다. 시료처리 24시간 후 PBS로 두 번 세척하여 1x lysis buffer로 용해한다. 상온에서 30분간 세포를 용해시킨 후, 4℃ 12000 rpm에서 15 분간 원심분리 한다. The experiment on the protein expression change of skin keratinocytes for the extract of ItBody Dolkim was carried out as follows. The cells are evenly distributed in a 60mm dish into 3.5×10 5 cells and then stabilized for 24 hours. Depletion and sample treatment after 24 hours. After 24 hours of sample treatment, wash twice with PBS and dissolve in 1x lysis buffer. After lysing the cells for 30 minutes at room temperature, centrifuge for 15 minutes at 12000 rpm at 4°C.
원심분리 후 상층액만 모아 -20 ℃에서 보관하여 사용한다. 단백질분석기법 (Immunoblotting) 을 위해 단백질 25㎍/lane을 10% SDS-Polyacrylamide gel 상에서 전기영동하고, membrane은 5% Skim milk를 첨가하여 상온에서 1시간 반응시킨다. 1차 항체 는 Tris Buffered saline에 1:1000 비율로 처리하였으며, 다음날 TBST (Tris Buffered saline-Tween 20)에 세 번 washing 후 2차 항체는 3% skim milk에 anti-rabbit 또는 anti-mouse antibody (2차 항체) 를 1:1000 비율로 1 시간 처리한 후 immunoblotting 한다.After centrifugation, only the supernatant is collected and stored at -20 ℃ for use. For protein analysis (Immunoblotting), 25㎍/lane of protein was electrophoresed on 10% SDS-Polyacrylamide gel, and 5% Skim milk was added to the membrane and reacted at room temperature for 1 hour. The primary antibody was treated with Tris Buffered saline at a ratio of 1:1000, and after washing three times in TBST (Tris Buffered saline-Tween 20) the next day, the secondary antibody was anti-rabbit or anti-mouse antibody (2) in 3% skim milk. Primary antibody) at a ratio of 1:1000 for 1 hour and then immunoblotting.
도 4는 잇바디 돌김 단일 화합물에 대한 피부각질세포의 단백질 발현의 변화를 나타낸 그림이다. 피부각질세포에 양성대조군으로 EGF10ng/ml, 잇바디 돌김 단일 화합물 P-334, Dehydrated. P-334를 1, 5, 10ug/ml로 처리한 결과 잇바디 돌김 단일 화합물이 양성대조군인 EGF10ng/ml 만큼 세포생장에 관여하는 P-ERK의 단백질 발현이 농도별로 증가하였다.4 is a diagram showing the change in protein expression of keratinocytes for a single compound of itbody protrusion. As a positive control for skin keratinocytes, EGF10ng/ml, itbody protrusion single compound P-334, Dehydrated. As a result of treatment with 1, 5, and 10 ug/ml of P-334, the protein expression of P-ERK involved in cell growth increased by concentration as much as EGF10 ng/ml, which is a positive control, in the Itbody protrusion single compound.
또한 잇바디 돌김 단일 화합물 P-334, Dehydrated.P-334 는 상피세포 성장 호르몬을 감지하는 수용체인 EGFR (Epidermal Growth Factor Receptor, 상피세포 성장 인자 수용체) 의 단백질 발현이 잇바디 돌김 단일 화합물 P-334에서는 변화가 없었으며, Dehydrated.P-334에서 농도별로 증가하였다. In addition, the protein expression of EGFR (Epidermal Growth Factor Receptor), a receptor that detects epithelial growth hormone, was changed in the IBD single compound P-334 and Dehydrated.P-334. There was no, and it increased by concentration in Dehydrated.P-334.
4. RT-PCR을 통한 피부재생 인자와 관련된 유전자 발현 확인4. Confirmation of gene expression related to skin regeneration factor through RT-PCR
RT-PCR을 통한 피부재생 인자와 관련된 유전자 발현 확인을 위해 피부각질세포인 HACAT 세포를 60mm plate에 2×105cell/ml의 세포 수로 분주하고 37℃, 5% CO2 조건에서 24시간 동안 배양하였다. EGF 10ng/ml을 전처리 한 후, 잇바디 돌김 단일 화합물 Dehydrated.P-334 1, 5, 10ug/ml을 처리하였다. 시료 처리 24시간 후, PBS (phosphate buffered saline)로 세척 한 후에 0.05% Trypsin-EDTA와 원심분리기를 이용하여 연골세포를 회수하였다. To confirm the expression of genes related to skin regeneration factors through RT-PCR, cutaneous keratinocytes, HACAT cells, were dispensed into a 60mm plate at a cell number of 2×10 5 cells/ml, and cultured for 24 hours at 37°C and 5% CO 2 I did. After pre-treatment of 10 ng/ml of EGF, it was treated with 1, 5, and 10 ug/ml of the single compound Dehydrated.P-334. After 24 hours of sample treatment, after washing with PBS (phosphate buffered saline), chondrocytes were recovered using 0.05% Trypsin-EDTA and a centrifuge.
회수된 세포를 RNeasy Mini Kit (Qiagen)를 사용하여 RNA를 분리하였다. 분리된 RNA를 이용하여 정량 후 cDNA를 합성하여 각각의 primer, DEPC water 그리고 TaKaRa Ex Taq (TaKaRa)을 이용하여 증폭하였다. 표 1은 잇바디 돌김 단일 화합물(P334, DP334)에 대한 피부각질세포의 mRNA의 발현을 위한 프라이머를 나타낸다. RNA was isolated from the recovered cells using RNeasy Mini Kit (Qiagen). After quantification using the separated 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 the mRNA of keratinocytes for the single compound of itbody protrusion (P334, DP334).
| EGFREGFR | sense: 5'-CTTGCAGCGATACAGCTCAG-3'antisense: 5-GGCTGTCGAATGTGCTGTTTG-3'sense: 5'-CTTGCAGCGATACAGCTCAG-3' antisense: 5-GGCTGTCGAATGTGCTGTTTG-3' |
| Beta-actinBeta-actin | sense: 5'-AGCGAGCATCCCCCAAAGTT-3'antisense: 5'-GGGCACGAAGGCTCATCATT-3'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-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
각 PCR 산물들의 양적 차이를 확인하기 위하여 1×TAE buffer로 2% agarose gel을 만들고 well 당 각각의 primer에 해당하는 PCR 산물에 DNA gel loading solution을 섞어서 loading 한 후 100 V에서 전기영동을 하였다. EtBr 을 이용하여 염색 한 후 UV 하에서 발현의 변화 여부를 확인하였다. 이때 housekeeping 유전자인 Beta-actin 을 internal control로 사용하였다. To check the quantitative difference of each PCR product, a 2% agarose gel was made with 1×TAE buffer, and a DNA gel loading solution was mixed and loaded with the PCR product corresponding to each primer per well, followed by electrophoresis at 100 V. After staining with EtBr, it was confirmed whether the expression was changed under UV. At this time, Beta-actin, a housekeeping gene, was used as an internal control.
도 5은 잇바디 돌김 단일 화합물(P334, DP334)에 대한 피부각질세포의 mRNA의 발현의 변화를 나타낸 그림이다. 피부재생세포(HACAT)에 성장인자인 EGF 10ng/ml 처리만큼 잇바디 돌김 화합물 Dehydrated.P-334 1, 5, 10ug/ml가 농도별로 증가하는 경향을 보였다. 잇바디 돌김 단일 화합물 Dehydrated.P-334 의 EGFR의 mRNA 발현이 양성대조군인 EGF 10ng/ml 만큼 농도별로 증가하였다. Fig. 5 is a diagram showing changes in the expression of mRNA in keratinocytes for the single compound of itbody protrusion (P334, DP334). As the growth factor EGF 10ng/ml was treated in skin regeneration cells (HACAT), the itbody protrusion compound Dehydrated.P-334 1, 5, 10ug/ml showed a tendency to increase by concentration. The mRNA expression of EGFR of Dehydrated.P-334, a single compound of itbody protrusion, increased by concentration by 10 ng/ml of EGF, a positive control.
본 발명은 김에서 분리한 단일 화합물들을 유효성분으로 함유하는 피부재생용 약학적 조성물 및 기능성 화장료 조성물을 추출함으로써 피부재생에 관여하는 유효성분 DP-334의 함량을 증가시켜 피부각질세포 (HACAT Cell)에서의 피부재생 효과와 피부 재생과 관련된 제품 생산 및 관련 사업자들의 이윤을 창출할 수 있어 산업상 이용가능성이 있다.The present invention extracts a pharmaceutical composition for skin regeneration and a functional cosmetic composition containing a single compound isolated from laver as an active ingredient, thereby increasing the content of DP-334, an active ingredient involved in skin regeneration, so that skin keratinocytes (HACAT Cell) It has industrial applicability as it can produce skin regeneration effects and profits for products related to skin regeneration and related businesses.
Claims (5)
- 잇바디 돌김(Porphyra. dentata) 열수 추출물을 유효성분으로 포함하는 것을 특징으로 하는 피부재생용 약학적 조성물 및 기능성 화장료 조성물A pharmaceutical composition for skin regeneration and a functional cosmetic composition comprising a hot water extract of Porphyra. dentata as an active ingredient
- 제1항에 있어서 잇바디 돌김 열수 추출물은 단일 화합물 포르피라-334(Porphyra-334) 또는 단일 화합물 Dehydrated P334를 포함하는 것을 특징으로 하는 피부재생용 약학적 조성물 및 기능성 화장료 조성물The pharmaceutical composition and functional cosmetic composition for skin regeneration according to claim 1, characterized in that the hot water extract of itbody dolgi contains a single compound Porphyra-334 or a single compound Dehydrated P334.
- 제1항에 있어서 잇바디 돌김 추출물에서 P-334(Porphyra-334)는 추출과정을 통하여 DP-334 (Dehydrated.Porphyra-334)로 구조적으로 변화되는 것인 피부 재생용 약학적 조성물 및 기능성 화장료 조성물The pharmaceutical composition and functional cosmetic composition for skin regeneration according to claim 1, wherein P-334 (Porphyra-334) is structurally changed to DP-334 (Dehydrated.Porphyra-334) through the extraction process
- 제1항에 있어서, 상기 조성물은 피부 세포 재생 촉진, 상처 치유 촉진의 효능을 가지는 것을 특징으로 하는 피부재생용 약학적 조성물 및 기능성 화장료 조성물The pharmaceutical composition and functional cosmetic composition for skin regeneration according to claim 1, wherein the composition has an effect of promoting skin cell regeneration and promoting wound healing.
- 제1항에 있어서 상기 조성물은 건조 중량에 대하여 0.001~2000 mg/kg 중량의 양을 함유하며, 산제, 과립제, 정제, 캡슐제, 에멀젼, 시럽, 경피제, 좌제 또는 멸균용 주사용, 밴드, 연고로 제형화되는 것을 특징으로 하는 피부재생용 약학적 조성물 및 기능성 화장료 조성물The method of claim 1, wherein the composition contains 0.001 to 2000 mg/kg weight based on dry weight, and includes powders, granules, tablets, capsules, emulsions, syrups, transdermals, suppositories or sterilization injections, bands, Pharmaceutical composition and functional cosmetic composition for skin regeneration, characterized in that formulated as an ointment
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