KR102206085B1 - Composition for preventing or improving skin photoaging comprising unsaponifiable matter from Perilla frutescens by-product as effective component - Google Patents
Composition for preventing or improving skin photoaging comprising unsaponifiable matter from Perilla frutescens by-product as effective component Download PDFInfo
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Abstract
본 발명은 들깨박 유래 비검화물(unsaponifiable matter)을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 화장료 조성물, 상기 화장료 조성물을 포함하는 자외선 차단제 및 들깨박 유래 비검화물을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 피부 외용제 조성물에 관한 것이다.The present invention is a cosmetic composition for preventing or improving skin photoaging containing unsaponifiable matter derived from perilla seed as an active ingredient, a sunscreen comprising the cosmetic composition, and preventing skin photoaging containing unsaponifiable matter derived from perilla seed as an active ingredient Or it relates to an external composition for skin improvement.
Description
본 발명은 들깨박 유래 비검화물을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition for preventing or improving skin photoaging, containing an unsaponifiable product derived from perilla seeds as an active ingredient.
광노화(photoaging)는 태양광선의 자외선(UVR, ultraviolet radiation)에 반복적이거나 장기간 노출되어 피부의 탄력 및 수분이 감소되고 주름이 형성되는 등의 피부손상이 일어나는 현상을 말한다. 생활수준의 향상으로 인해 아름답고 건강한 피부를 유지하려는 욕구가 증대되고, 야외활동이 증가하면서 태양광선 노출로부터 야기되는 피부손상을 방지하기 위한 관심이 증가함에 따라 피부노화 억제에 관한 연구가 활발히 진행되고 있다. Photoaging refers to a phenomenon in which skin damage occurs, such as decreased elasticity and moisture of the skin and wrinkles formed due to repeated or prolonged exposure to ultraviolet radiation (UVR) of sunlight. As the desire to maintain beautiful and healthy skin increases due to the improvement of the standard of living, and as interest in preventing skin damage caused by exposure to sunlight increases as outdoor activities increase, research on skin aging control is being actively conducted. .
자외선은 파장에 따라 UVA(ultraviolet A, 320~400nm), UVB(ultraviolet B, 280~320nm), UVC(ultraviolet C, 200~280nm)로 분류된다. 그 중 UVB는 매우 유해한 광선으로 표피 손상에 관여하거나 건조, 피부홍반, 색소침착, 표피비후(epidermal thickening) 등을 유발한다. 또한 항산화 기전의 불균형을 유발하여 피부를 구성하는 지질, 단백질, 효소 등을 손상시킴으로써 염증반응과 피부암, 광노화를 심화시킨다. 반면, UVA는 UVB에 비해 단기간의 위력은 미미하지만 파장이 길어서 피부 깊숙이 침투해 피부의 진피를 손상시켜서 피부를 검게 하고 피부노화를 촉진시킨다. UVA 및 UVB를 저해할 수 있는 소재 및 제품의 효능기준이 자외선 차단지수로 이용되고 있으며, 피부 광노화에 따른 피부손상을 위해 장기간 사용시에도 부작용없이 효능을 나타낼 수 있는 천연 기능성 소재 및 제품을 개발하기 위한 연구가 활발히 이루어지고 있다.UV rays are classified into UVA (ultraviolet A, 320-400nm), UVB (ultraviolet B, 280-320nm), and UVC (ultraviolet C, 200-280nm) according to their wavelength. Among them, UVB is very harmful rays that are involved in epidermal damage or cause dryness, skin erythema, pigmentation, and epidermal thickening. In addition, it causes an imbalance of antioxidant mechanisms and damages lipids, proteins, and enzymes that make up the skin, thereby intensifying inflammatory reactions, skin cancer, and photoaging. On the other hand, UVA has little power in a short term compared to UVB, but its long wavelength penetrates deep into the skin and damages the dermis of the skin, blackening the skin and promoting skin aging. The standard of efficacy of materials and products that can inhibit UVA and UVB is used as the UV blocking index, and to develop natural functional materials and products that can exhibit efficacy without side effects even when used for a long time for skin damage due to skin photoaging. Research is being actively conducted.
UVB 자극에 의해 생성되는 ROS(reactive oxygen species)는 광노화의 원인 중 하나로 세포 내 신호전달체계를 자극하여 DNA, 지질 및 단백질 등 생체분자에 산화적 스트레스 유발을 통한 피부조직 손상을 일으킨다. ROS는 표피 각질세포와 진피 섬유아세포의 자극으로 AP-1 및 NF-κB(nuclear factor kappa-light-chain-enhancer of activator B cells)의 전사인자를 활성화시키고 염증관련 사이토카인(cytokine)을 분비하도록 하여 MMP(matrix metalloproteinase)의 발현을 증가시키고 피부조직을 구성하는 교원질과 탄력섬유의 분해를 촉진하여 주름을 형성시킨다. 생체에서는 ROS 자극으로부터 생성 및 활성되는 지질과산화물과 산화효소로부터 방어하기 위해 CAT(catalase), SOD(superoxide dismutase) 및 GST(glutathione-s-transferase) 등의 항산화 효소가 작용하며, 체외에서는 비타민 C, E 및 폴리페놀류와 플라보노이드 등의 생리활성물질을 통해 ROS에 의한 손상을 방어한다.ROS (reactive oxygen species) produced by UVB stimulation is one of the causes of photoaging and stimulates the intracellular signaling system, causing oxidative stress on biomolecules such as DNA, lipids and proteins, causing skin tissue damage. ROS stimulates epidermal keratinocytes and dermal fibroblasts to activate transcription factors of AP-1 and nuclear factor kappa-light-chain-enhancer of activator B cells (NF-κB) and secrete inflammation-related cytokines. Thus, it increases the expression of MMP (matrix metalloproteinase) and promotes the decomposition of collagen and elastic fibers constituting the skin tissue to form wrinkles. In vivo, antioxidant enzymes such as CAT (catalase), SOD (superoxide dismutase) and GST (glutathione-s-transferase) act to protect against lipid peroxides and oxidative enzymes produced and activated from ROS stimulation. In vitro, vitamin C, It protects against damage caused by ROS through bioactive substances such as E and polyphenols and flavonoids.
들깨(Perilla frutescens)는 쌍떡잎식물 통화식물목 꿀풀과(Labiatae, mint family)의 한해살이풀로, 안토시아닌(anthocyanin)의 유효성에 따라 붉은색의 색소가 있는 형태와 색소가 없는 녹색 형태의 물질대사적 표현형(metabolic phenotypes)의 2가지 화학변종 형태(chemovarietal forms)가 존재한다. 들깨는 전통적인 약용작물 중의 하나로 우울증, 종양, 기침, 항균, 항곰팡이, 알려지 등의 다양한 질병을 다스리는데 사용되어 왔다. Perilla frutescens ) is an annual plant of the labiatae, mint family, a dicotyledonous plant, and metabolic phenotypes in a reddish pigmented form and a greenish pigmented form depending on the effectiveness of anthocyanins. There are two chemovarietal forms. Perilla seeds are one of the traditional medicinal crops, and have been used to treat various diseases such as depression, tumors, cough, antibacterial, antifungal, and known.
한편, 한국등록특허 제1934793호에는 '고욤나무 잎 추출물을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 조성물'이 개시되어 있고, 한국등록특허 제0614970호에는 들깨박 추출물을 유효성분으로 함유하는 인지기능장애 예방 및 개선용 조성물'이 개시되어 있으나, 본 발명의 들깨박 유래 비검화물을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 화장료 조성물에 대해서는 기재된 바가 없다.On the other hand, Korean Patent No. 1934793 discloses'a composition for preventing or improving skin photoaging that contains an extract of Goyom trees as an active ingredient', and Korean Patent No. 0614970 discloses a recognition containing perilla seed extract as an active ingredient. A composition for preventing and improving dysfunction is disclosed, but there is no description of a cosmetic composition for preventing or improving skin photoaging containing the non-saponifiable product derived from perilla seeds of the present invention as an active ingredient.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 인간 섬유아세포주 Hs68 세포에 UVB를 조사하여 광노화를 유도한 후 들깨박 유래 비검화물을 처리한 결과, UVB 조사에 의해 감소된 Hs68 세포의 생존율이 증가하고 UVB에 의해 증가된 ROS 생성량은 감소하는 것을 확인하였다. 또한, 본 발명의 들깨박 유래 비검화물은 UVB 조사에 의해 유도되는 MAPKs(JNK, ERK 및 p38) 및 AP-1(c-fos 및 c-jun)의 인산화 반응을 억제하여 피부 노화를 유도하는 MMP 단백질의 발현을 감소시키고 콜라겐 생성을 촉진하는 TGF(transforming growth factor)-β1 및 Smad2/3의 발현을 증가시키는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above requirements, and the present inventors treated human fibroblast cell line Hs68 cells with UVB to induce photoaging and then treated unsaponifiable products derived from perilla seeds, resulting in reduced Hs68 cells by UVB irradiation. It was confirmed that the survival rate of was increased and the amount of ROS production increased by UVB decreased. In addition, the unsaponifiable product derived from perilla seeds of the present invention inhibits the phosphorylation of MAPKs (JNK, ERK and p38) and AP-1 (c-fos and c-jun) induced by UVB irradiation, thereby inducing skin aging. The present invention was completed by confirming that the expression of the transforming growth factor (TGF)-β1 and Smad2/3, which decreases protein expression and promotes collagen production, is increased.
상기 과제를 해결하기 위해, 본 발명은 들깨박 유래 비검화물(unsaponifiable matter)을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 화장료 조성물을 제공한다.In order to solve the above problems, the present invention provides a cosmetic composition for preventing or improving skin photoaging, containing an unsaponifiable matter derived from perilla seed as an active ingredient.
또한, 본 발명은 상기 화장료 조성물을 포함하는 자외선 차단제를 제공한다.In addition, the present invention provides a sunscreen comprising the cosmetic composition.
또한, 본 발명은 들깨박 유래 비검화물(unsaponifiable matter)을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 피부 외용제 조성물을 제공한다.In addition, the present invention provides a composition for external application for skin for preventing or improving skin photoaging, containing unsaponifiable matter derived from perilla seeds as an active ingredient.
본 발명의 들깨박 유래 비검화물은 피부노화를 유도하는 단백질들의 활성을 감소시킬 뿐만 아니라 콜라겐 생성을 촉진하는 인자들의 활성을 증가시키는 효과가 우수하므로, 자외선에 의한 광노화를 예방 및 개선하기 위한 기능성 소재 및 화장료 조성물로 유용하게 사용될 수 있다.The unsaponifiable material derived from perilla seeds of the present invention not only reduces the activity of proteins that induce skin aging, but also has an excellent effect of increasing the activity of factors that promote collagen production, so it is a functional material for preventing and improving photoaging caused by ultraviolet rays. And it can be usefully used as a cosmetic composition.
도 1은 인간 섬유아세포주인 Hs68 세포에 들깨박 비검화물(PSU) 또는 들깨박 메탄올 추출물(1, 2.5 또는 5㎍/㎖)을 전처리한 후 UVB를 조사하여 세포의 생존율을 나타낸 그래프(A)와 Hs68 세포에 PSU를 전처리한 후 UVB를 조사하여 세포의 생존율(B 및 C) 및 세포 내 콜라겐 생성량(D 및 E)을 나타낸 그래프이다.
도 2는 Hs68 세포에 PSU(1, 2.5 또는 5㎍/㎖)를 전처리한 후 UVB를 조사하여 세포 내에서 생성되는 ROS(reactive oxygen species)를 형광 분광기(fluorescent spectrophotometer)로 측정하여 나타낸 그래프이다.
도 3은 Hs68 세포에 PSU(1, 2.5 또는 5㎍/㎖)를 전처리한 후 UVB를 조사하여 MMP(matrix metalloproteinase)-1 및 MMP-3 단백질의 생성량(A 및 B) 및 mRNA 발현량(C 및 D)을 측정하여 나타낸 그래프이다.
도 4는 Hs68 세포에 PSU(1, 2.5 또는 5㎍/㎖)를 전처리한 후 UVB를 조사하여 JNK(A), ERK(B) 및 p38(C)의 인산화 수준을 측정한 결과이다.
도 5는 Hs68 세포에 PSU(1, 2.5 또는 5㎍/㎖)를 전처리한 후 UVB를 조사하여 c-jun(A) 및 c-fos(B)의 인산화 활성을 측정한 결과이다.
도 6은 Hs68 세포에 PSU(1, 2.5 또는 5㎍/㎖)를 전처리한 후 UVB를 조사하여 TGF-β1(A), Smad2/3(B) 및 Smad7(C)의 발현량을 측정하여 나타낸 그래프이다.1 is a graph (A) showing the survival rate of cells by irradiation with UVB after pretreatment of perilla seed meal unsaponifiable (PSU) or perilla seed meal methanol extract (1, 2.5 or 5 μg/ml) to human fibroblast cell line Hs68 cells. A graph showing cell viability (B and C) and intracellular collagen production (D and E) by UVB irradiation after pre-treatment of Hs68 cells with PSU.
FIG. 2 is a graph showing the ROS (reactive oxygen species) generated in the cells after pretreatment with PSU (1, 2.5 or 5 μg/ml) on Hs68 cells and then irradiation with UVB, measured with a fluorescence spectrophotometer.
3 shows the production amount (A and B) and mRNA expression level (C) of MMP (matrix metalloproteinase)-1 and MMP-3 proteins by UVB irradiation after pretreatment of PSU (1, 2.5 or 5 μg/ml) in Hs68 cells. And D) are measured and shown.
4 is a result of measuring the phosphorylation levels of JNK(A), ERK(B) and p38(C) by pretreatment with PSU (1, 2.5 or 5 μg/ml) in Hs68 cells and irradiation with UVB.
5 is a result of measuring the phosphorylation activity of c-jun (A) and c-fos (B) by pretreatment with PSU (1, 2.5 or 5 μg/ml) in Hs68 cells and irradiation with UVB.
Figure 6 shows by measuring the expression levels of TGF-β1 (A), Smad2/3 (B) and Smad7 (C) by pretreatment with PSU (1, 2.5 or 5 μg/ml) in Hs68 cells and irradiation with UVB. It is a graph.
본 발명의 목적을 달성하기 위하여, 본 발명은 들깨박 유래 비검화물(unsaponifiable matter)을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 화장료 조성물을 제공한다.In order to achieve the object of the present invention, the present invention provides a cosmetic composition for preventing or improving skin photoaging, containing as an active ingredient unsaponifiable matter derived from perilla.
본 명세서에서, 용어 '비검화물'은 불검화물이라고도 하며, 일반적으로 알칼리에 의해 가수분해되지 않는 기질을 의미하는 것으로서, 지질 중 검화물과 비교하였을 때 스테롤류 등의 탄화수소, 지용성 색소 등 비타민류 등을 포함하는 물질을 의미한다.In the present specification, the term'unsaponifiable' is also referred to as unsaponifiable, and generally refers to a substrate that is not hydrolyzed by alkali, and when compared to saponified in lipids, hydrocarbons such as sterols, vitamins such as fat-soluble pigments, etc. It means a material containing.
본 발명의 화장료 조성물에 있어서, 들깨박 유래 비검화물은 당업계에 알려진 통상의 방법을 이용하여 제조할 수 있고, 바람직하게는 들기름을 제조한 후 얻어진 들깨 부산물인 들깨박에 알칼리를 처리하여 검화물을 분해하여 수득할 수 있다. 이때, 분해된 검화물을 제외하고 남은 잔여물에 유기용매를 이용하여 추출할 수 있으나, 이에 제한되지 않는다. 상기 유기용매는 헥산, 에틸아세테이트 또는 이들의 혼합 용액일 수 있고, 보다 바람직하게는 헥산과 에틸아세테이트의 혼합 용액일 수 있으나, 이에 제한되지 않는다.In the cosmetic composition of the present invention, the unsaponifiable product derived from perilla seed can be prepared using a conventional method known in the art, and preferably, a saponified product is treated with an alkali to the perilla by-product obtained after preparing perilla oil. Can be obtained by decomposition. At this time, the remaining residue except for the decomposed saponified product may be extracted using an organic solvent, but is not limited thereto. The organic solvent may be hexane, ethyl acetate, or a mixed solution thereof, more preferably a mixed solution of hexane and ethyl acetate, but is not limited thereto.
본 명세서에서, 용어 '광노화'는 태양광선의 자외선에 반복적이거나 장기간 노출시 피부손상, 색소침착, 주름생성 또는 피부의 탄력이 저하되는 현상을 말한다.In the present specification, the term'photoaging' refers to a phenomenon in which skin damage, pigmentation, wrinkle formation, or skin elasticity decreases upon repeated or prolonged exposure to ultraviolet rays of sunlight.
본 발명의 화장료 조성물에 있어서, 상기 들깨박 유래 비검화물은 콜라겐 합성을 증가시키고 콜라겐분해효소인 MMP(matrix metalloproteinase)-1 또는 MMP-3의 발현을 감소시키는 효과가 우수하다. 들깨박 비검화물을 이용하여 type Ⅰ 콜라겐의 분해효소인 MMP-1 단백질과 type Ⅳ 콜라겐의 분해효소인 MMP-3 단백질의 활성을 억제시키면, 콜라겐 분해를 감소시킬 수 있으므로 피부조직의 탄력을 유지하여 주름 생성을 예방할 수 있다.In the cosmetic composition of the present invention, the unsaponifiable product derived from perilla seeds has an excellent effect of increasing collagen synthesis and reducing the expression of collagen-degrading enzymes MMP (matrix metalloproteinase)-1 or MMP-3. Inhibiting the activity of MMP-1 protein, type I collagen degrading enzyme and MMP-3 protein, type IV collagen degrading enzyme using unsaponifiable perilla, can reduce collagen degradation, thus maintaining elasticity of skin tissue. It can prevent wrinkles.
또한, 상기 들깨박 유래 비검화물은 세포 내의 ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), p38, c-fos 또는 c-jun의 인산화 반응을 감소시키고, TGF-β1 및 Smad2/3의 발현을 증가시켜 MMPs 단백질의 발현을 감소시켜 피부 노화를 방지하는 효과가 우수하다. MAPK(mitogen activated protein kinase; ERK, JNK 및 p38) 경로의 활성화는 AP-1(Fos 및 Jun family 단백질로 이루어진 전사인자)의 연속적인 활성을 유도하여 MMPs의 발현을 증가시킬 수 있다. 예를 들어, ERK의 인산화는 AP-1을 구성하는 c-fos의 발현 및 인산화를 증가시키고, JNK 또는 p38의 인산화는 AP-1을 구성하는 c-jun의 발현 및 인산화를 증가시켜 MMPs의 발현을 증가시킨다. 따라서, 들깨박 추출물을 이용하여 MAPK 및 AP-1의 활성을 저해하면 MMPs의 발현을 억제시켜 주름 생성을 예방할 수 있다. 또한, 들깨박 추출물을 이용하여 TGF(transforming growth factor)-β1 및 Smad2/3의 발현 및 인산화를 유도하여 활성을 증가시키면 핵 내의 전사인자의 활성화를 통해 콜라겐의 생성을 촉진시킬 수 있다.In addition, the unsaponifiable product derived from perilla seeds decreases the phosphorylation reaction of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, c-fos or c-jun in cells, and TGF-β1 And it is excellent in the effect of preventing skin aging by increasing the expression of Smad2/3 to decrease the expression of the MMPs protein. Activation of the mitogen activated protein kinase (MAPK; ERK, JNK, and p38) pathway can increase the expression of MMPs by inducing the continuous activity of AP-1 (a transcription factor consisting of Fos and Jun family proteins). For example, phosphorylation of ERK increases the expression and phosphorylation of c-fos constituting AP-1, and phosphorylation of JNK or p38 increases the expression and phosphorylation of c-jun constituting AP-1 to express MMPs. Increase Therefore, by inhibiting the activity of MAPK and AP-1 using perilla extract, it is possible to prevent wrinkles by inhibiting the expression of MMPs. In addition, if the perilla extract is used to induce the expression and phosphorylation of TGF (transforming growth factor)-β1 and Smad2/3 to increase the activity, it is possible to promote collagen production through activation of transcription factors in the nucleus.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있고, 보다 상세하게는, 스킨, 스킨 소프트너, 스킨 토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐로션, 영양로션, 마사지크림, 영양크림, 아이크림, 모이스쳐크림, 자외선 차단제, 핸드크림, 에센스, 영양에센스, 팩, 클렌징폼, 클렌징워터, 클렌징크림, 바디로션, 바디클렌져, 비누 및 파우더 중에서 선택된 어느 하나의 제형으로 제조될 수 있으며, 바람직하게는 자외선 차단제 형태로 제조될 수 있으나, 이에 제한되지 않는다. 이들 각 제형으로 이루어진 화장료 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다. The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion It can be formulated as a foundation, wax foundation and spray, and more specifically, skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, Moisture cream, sunscreen, hand cream, essence, nutritional essence, pack, cleansing foam, cleansing water, cleansing cream, body lotion, body cleanser, soap, and powder can be prepared in any one formulation selected from, preferably UV It may be prepared in the form of a blocking agent, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.
본 발명의 화장료 조성물에는 상기 유효성분 이외에 화장품 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온 봉쇄제 및 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭과 같은 통상적인 보조제, 그리고 담체를 포함한다.In addition to the above active ingredients, the cosmetic composition of the present invention includes ingredients commonly used in cosmetic compositions, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents ( foaming agents), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives , Conventional adjuvants such as lipid vesicles, and carriers.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as carrier components Etc. may be used.
본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판-부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydro Propellants such as carbon, propane-butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, a solvating agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.
본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 아세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 리놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a surfactant containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acetionate, imidazolinium derivative, methyltaurate, sarcosinate , Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty diethanolamide, vegetable oil, linoline derivative or ethoxylated glycerol fatty acid ester, and the like may be used.
본 발명의 화장료 조성물은 형광물질, 살진균제, 굴수성 유발물질, 보습제, 방향제, 방향제 담체, 단백질, 용해화제, 당 유도체, 일광차단제, 비타민, 식물 추출물 등을 포함하는 부형제를 추가로 함유할 수 있다.The cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrophobic substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreen agents, vitamins, plant extracts, etc. .
본 발명의 화장료 조성물은 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 성분이나, 효과를 증강하는 성분을 1종 이상 함유할 수 있다. 예컨대, 당업계에 공지된 피부 노화방지 및 주름개선 성분으로서, 레티노산, 동물 태반 유래의 단백질, 베튤린산 및 클로렐라 추출물로부터 선택되는 1종 또는 2종 이상의 성분을 추가로 포함할 수 있다. 상기 추가된 성분은 전체 조성물 100 중량부에 대하여, 0.0001 내지 10 중량부로 포함될 수 있을 것이며, 상기 함량 범위는 콜라겐 합성 촉진 활성, 피부 안전성, 제형화 시의 용이성 등의 요건에 따라 조절될 수 있을 것이다.In addition to the active ingredient, the cosmetic composition of the present invention may further contain one or more ingredients that exhibit the same or similar functions or enhance the effect. For example, as an anti-aging and wrinkle-improving component known in the art, one or more components selected from retinoic acid, protein derived from animal placenta, betulinic acid and chlorella extract may be further included. The added component may be included in an amount of 0.0001 to 10 parts by weight, based on 100 parts by weight of the total composition, and the content range may be adjusted according to requirements such as collagen synthesis promoting activity, skin safety, and ease of formulation. .
본 발명은 또한, 들깨박 유래 비검화물을 유효성분으로 함유하는 피부 광노화 예방 또는 개선용 피부 외용제 조성물을 제공한다.The present invention also provides a composition for external application for skin for preventing or improving skin photoaging, containing an unsaponifiable product derived from perilla seed as an active ingredient.
본 발명의 피부 광노화 예방 또는 개선용 피부 외용제 조성물에 있어서, 상기 들깨박 유래 비검물은 콜라겐 합성을 증가시키고 콜라겐분해효소인 MMP(matrix metalloproteinase)-1 또는 MMP-3의 발현을 감소시킬 뿐만 아니라, 세포 내의 ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), p38, c-fos 또는 c-jun의 인산화 반응을 감소시키고, TGF-β1 및 Smad2/3의 발현을 증가시켜 콜라겐분해효소인 MMPs의 활성을 감소시킬 수 있으므로, 자외선에 반복적이거나 장기간 노출시 유발된 피부손상, 색소침착, 주름생성 또는 피부 탄력 저하와 관련 광노화를 방지하는 효과가 우수하다.In the composition for external application for skin for preventing or improving skin photoaging of the present invention, the non-sample derived from perilla seeds not only increases collagen synthesis and decreases the expression of collagenase, matrix metalloproteinase (MMP)-1 or MMP-3, By reducing the phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), p38, c-fos or c-jun in cells, and increasing the expression of TGF-β1 and Smad2/3 Since it can reduce the activity of MMPs, which are collagenases, it is excellent in preventing skin damage, pigmentation, wrinkle formation, or reduction of skin elasticity and related photoaging caused by repeated or prolonged exposure to UV rays.
본 발명의 피부 외용제는 본 발명의 조성물을 다양한 방식 또는 형태로 인체에 적용할 수 있으나, 바람직하게는 피부에 국소적으로 도포되어 적용 가능한 피부 외용제의 제형으로 제조될 수 있다. 예를 들어, 파우더, 연고제, 겔제, 크림제, 리니멘트제, 로션제, 액제, 또는 에어로졸제 등을 들 수 있으나, 이에 한정되는 것은 아니다.The external preparation for skin of the present invention may be applied to the human body in various ways or forms of the composition of the present invention, but may be preferably prepared in a formulation of a skin external preparation applicable by being applied topically to the skin. For example, a powder, an ointment, a gel, a cream, a liniment, a lotion, a liquid, or an aerosol may be used, but the present invention is not limited thereto.
상기 피부 외용제로 제제화할 경우, 본 발명의 조성물 외에 첨가물 등을 적절하게 배합하여 제조할 수 있다. 상기 첨가물은 당 업계에서 통상적으로 이용되는 것으로, 본 발명의 목적 및 효과를 손상시키지 않는 범위 내에서 배합 가능하다.When formulated for the external application for skin, it can be prepared by appropriately blending additives in addition to the composition of the present invention. The additives are commonly used in the art and can be blended within a range that does not impair the objects and effects of the present invention.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
재료 및 방법Materials and methods
1. One. 들깨박Perilla 유래 origin 비검화물의Unsaponifiable 제조 Produce
제작용기에 들기름을 제조하고 남은 들깨박(들깨 부산물) 3g을 취하여 6% 피로갈롤(pyrogallol) 20㎖을 첨가한 후 질소 충진하고 5분 동안 초음파 처리하고 수산화칼륨(KOH) 8㎖을 첨가하였다. 다시 질소 충진하고 뚜껑을 냉각관으로 교체해주었으며, 75℃의 항온기에서 50분간 반응시킨 후 냉각하였다. 냉각 후 2% 염화나트륨(NaCl) 30㎖을 첨가하고 추출용매(hexane:ethyl acetate=85:15, without BHT) 30㎖씩로 3번 반복 추출하였다. 상기 추출물은 감압 농축하여 용매를 제거하고 DMSO(dimethyl sulfoxide)에 재용해하여 세포에 처리하였다.Perilla oil was prepared in a production container, and 3 g of the remaining perilla seed (perilla by-product) was taken, 20 ml of 6% pyrogallol was added, followed by nitrogen filling, ultrasonic treatment for 5 minutes, and potassium hydroxide (KOH) 8 ml. After filling with nitrogen again, the lid was replaced with a cooling tube, and the mixture was cooled after reacting for 50 minutes in a thermostat at 75°C. After cooling, 30 ml of 2% sodium chloride (NaCl) was added, and extraction was repeated 3 times with 30 ml of an extraction solvent (hexane:ethyl acetate=85:15, without BHT). The extract was concentrated under reduced pressure to remove the solvent and redissolved in dimethyl sulfoxide (DMSO) to treat cells.
2. 비타민 E 함량 측정2. Vitamin E content measurement
상기 제조된 들깨박 비검화물을 헥산(hexane)에 재용해하고 필터하여 NPHPLC(normal phase HPLC)로 분석하였다. LiChrosphere® Diol 100 컬럼(250 x 4 mm i.d., 5 um, Merck, 독일)을 이용하여 수행하였으며, 이동상은 헥산(hexane)/아이소프로판올(isopropanol)(98.9:1.1, v/v)을 1.0㎖/분의 속도로 흘려주었다. 비검화물의 토코페롤(tocopherol)과 토코트리에놀(tocotrienol)의 농도는 표준물질(standard)과 비교하여 평균 피크 면적을 사용하여 계산하였다.The prepared perilla meal unsaponifiable was redissolved in hexane, filtered, and analyzed by NPHPLC (normal phase HPLC). LiChrosphere ® Diol 100 column (250 x 4 mm id, 5 um, Merck, Germany) was used, and the mobile phase was 1.0 ml/ of hexane/isopropanol (98.9:1.1, v/v) It flowed at the speed of minutes. The concentrations of unsaponifiable tocopherol and tocotrienol were calculated using the average peak area compared to the standard.
3. 3. 피토스테롤Phytosterol (( phytosterolphytosterol ), ), 폴리코사놀Policosanol (( policosanolpolicosanol ), 스쿠알렌(), squalene ( squalene)의squalene) 분석 analysis
들깨박 비검화물 내의 피토스테롤, 폴리코사놀 및 스쿠알렌 농도는 5α-콜레스탄(cholestane)을 내부표준물질로 하여 측정하였다. 가스 크로마토그래피(Varian 3800; Varian Inc., 미국)에는 SAC-5 fused-silica capillary 컬럼(30m×0.32mm i.d.; Supelco, 미국)과 불꽃 이온화 검출기(flame ionization detector)가 장착되어 있다. 수송용 가스는 헬륨(helium)이고 주입기(injector)와 검출기(detector)의 온도는 각각 310℃와 320℃이며, 보유시간과 표준물질을 통해 피토스테롤, 폴리코사놀 및 스쿠알렌을 각각 구별하였다.The concentrations of phytosterol, policosanol, and squalene in the unsaponifiable perilla meal were measured using 5α-cholestane as an internal standard. Gas chromatography (Varian 3800; Varian Inc., USA) was equipped with a SAC-5 fused-silica capillary column (30m×0.32mm i.d.; Supelco, USA) and a flame ionization detector. The transport gas was helium, and the temperatures of the injector and detector were 310°C and 320°C, respectively, and phytosterol, policosanol, and squalene were distinguished through retention time and standard materials.
4. 세포배양 및 세포독성 실험4. Cell culture and cytotoxicity test
인간 섬유아세포주인 Hs68 세포(American Type Culture Collection, Manassas, 미국)는 FBS(fetal bovine serum, 10%)와 페니실린(penicillin, 100unit/㎖), 스트렙토마이신(streptomycin, 50㎍/㎖)이 함유된 DMEM 배지를 사용하여 37℃, 5% CO2, 95% 습공기(humid air)로 조절된 배양기에서 배양하였다. Hs68 세포는 96-웰 배양기에 1.0×104cells/well의 농도로 분주하여 24시간 동안 배양한 후 FBS가 포함되지 않은 배지에 시료를 희석하여 24시간 동안 배양하였다. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, 5mg/㎖) 용액 20㎕을 각 웰에 첨가하고 2시간 동안 반응시킨 후 생성된 청색 포르마잔(formazan) 결정을 DMSO로 용해시켜 흡광도를 측정하였다.Human fibroblast line Hs68 cells (American Type Culture Collection, Manassas, USA) are DMEM containing FBS (fetal bovine serum, 10%), penicillin (100 units/ml), and streptomycin (50µg/ml). The medium was cultured in an incubator controlled by 37° C., 5% CO 2 , and 95% humid air. Hs68 cells were dispensed in a 96-well incubator at a concentration of 1.0×10 4 cells/well and cultured for 24 hours, and then the samples were diluted in a medium containing no FBS and cultured for 24 hours. 20 µl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, 5 mg/ml) solution was added to each well and reacted for 2 hours, and the blue formazan ( formazan) crystals were dissolved in DMSO to measure absorbance.
5. 5. UVBUVB 조사에 대한 세포 보호 효과 Cellular protective effect on irradiation
Hs68 세포를 96-웰 배양기에 1.0×104cells/well의 농도로 분주하고 24시간 동안 배양한 후 FBS가 포함되지 않은 배지에 시료를 희석하여 24시간 동안 배양하였다. 배양이 끝난 후 배지를 제거하고 PBS(phosphate buffered saline)로 1회 세척한 다음 UVB 램프로 UVB(30mJ/cm2)를 조사하고, UV LIGHT METER(UV-340A, LT Lutron, 대만)를 사용하여 자외선 강도를 모니터링하였다. 모든 UVB 조사는 96-웰 배양기에 부착된 세포에 PBS를 얇게 도포한 후 수행되었으며, 조사 후 FBS가 함유되지 않은 배지에서 시료를 희석하고 24시간 동안 세포에 조사하였다. 대조군은 UVB 조사 없이 상기와 동일한 조건으로 진행하였으며, UVB에 대한 세포 보호 효과는 상기 MTT를 이용한 세포독성 실험과 동일하게 방법을 통해 확인하였다.Hs68 cells were dispensed in a 96-well incubator at a concentration of 1.0 × 10 4 cells/well, cultured for 24 hours, and then diluted in a medium containing no FBS and cultured for 24 hours. After the culture was completed, the medium was removed, washed once with PBS (phosphate buffered saline), and then irradiated with UVB (30mJ/cm 2 ) with a UVB lamp, and a UV LIGHT METER (UV-340A, LT Lutron, Taiwan) was used. UV intensity was monitored. All UVB irradiation was performed after applying a thin layer of PBS to the cells attached to the 96-well incubator, and after irradiation, the sample was diluted in a medium containing no FBS and irradiated to the cells for 24 hours. The control was carried out under the same conditions as above without UVB irradiation, and the cell protective effect against UVB was confirmed through the same method as in the cytotoxicity experiment using the MTT.
6. 6. ROSROS 생성량 측정 Measurement of production
Hs68 세포에 시료를 24시간 동안 전처리하고 PBS로 세척한 후 UVB(30mJ/cm2)를 세포에 조사하였다. UVB가 조사된 세포에 시료를 30분간 처리하고 25μM의 DCFH-DA(dichlorodihydro-fluorescein diacetate)로 염색한 후 형광 분광기(fluorescent spectrophotometer)를 이용하여 485nm의 여기 파장(excitation) 및 530nm의 방출 파장(emission)으로 세포 내 ROS(reactive oxygen species) 생성량을 측정하였다.Samples were pretreated to Hs68 cells for 24 hours, washed with PBS, and then UVB (30mJ/cm 2 ) was irradiated on the cells. The sample was treated on UVB-irradiated cells for 30 minutes, stained with 25 μM of DCFH-DA (dichlorodihydro-fluorescein diacetate), and then excitation wavelength of 485 nm and emission wavelength of 530 nm using a fluorescent spectrophotometer. ) To measure the amount of ROS (reactive oxygen species) produced in cells.
7. 수용성 콜라겐 및 7. Water-soluble collagen and MMPsMMPs 측정 Measure
MMP-1 및 MMP-3 생성량은 human MMP-1 및 MMP-3 ELISA 키트(Merck & Co. Inc., 미국)를 이용하여 배지에 존재하는 MMP-1 및 MMP-3의 함량을 측정하였고, 수용성 콜라겐의 함량은 SircolTM soluble collagen assay 키트를 사용하여 측정하였다.The amount of MMP-1 and MMP-3 produced was determined by measuring the content of MMP-1 and MMP-3 present in the medium using a human MMP-1 and MMP-3 ELISA kit (Merck & Co. Inc., USA). The content of collagen was measured using a Sircol TM soluble collagen assay kit.
8. 8. qRTqRT -- PCRPCR 을 이용한 Using mRNAmRNA 발현량 측정 Expression level measurement
총 RNA는 TRIzol reagent(SolGent)를 이용하여 추출하였고, cDNA는 (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific, Inc.)를 이용하여 합성하였다. 상기 합성된 cDNA, 프라이머, RNase-free water 등이 혼합된 반응 혼합액을 이용하여 qRT-PCR(quantitative real-time polymerase chain reaction) 수행하였으며, PCR에 사용된 TaqMan 프라이머는 Thermo Fisher Scientific에서 구입하여 사용하였다(MMP1 Assay ID; Hs00899658_m1, MMP3 Assay ID; Hs00968305_m1).Total RNA was extracted using TRIzol reagent (SolGent), and cDNA was synthesized using (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific, Inc.). QRT-PCR (quantitative real-time polymerase chain reaction) was performed using a reaction mixture in which the synthesized cDNA, primer, RNase-free water, etc. were mixed, and TaqMan primers used for PCR were purchased and used from Thermo Fisher Scientific. (MMP1 Assay ID; Hs00899658_m1, MMP3 Assay ID; Hs00968305_m1).
9. Western blot을 이용한 단백질 발현량 측정9. Measurement of protein expression level using Western blot
10% SDS-폴리아크릴아마이드 겔에 샘플을 로딩한 후 PVDF 멤브레인으로 트랜스퍼하였다. 멤브레인을 5% 탈지유가 함유된 TBST(Tris-Buffered Saline+Tween) 용액에서 1시간 동안 블로킹한 후 5% 탈지유가 함유된 TBST 용액으로 희석한 1차 항체를 이용하여 4℃에서 밤새 반응시켰다. 1차 항체 반응이 끝난 후 TBST 용액으로 30분간 세척한 후 2차 항체를 이용하여 반응시킨 다음 암실에서 ECL 용액을 처리하여 X-ray 필름에 감광시켰다.After loading the sample onto a 10% SDS-polyacrylamide gel, it was transferred to a PVDF membrane. The membrane was blocked in a TBST (Tris-Buffered Saline+Tween) solution containing 5% skim milk for 1 hour, and then reacted overnight at 4°C using a primary antibody diluted with a TBST solution containing 5% skim milk. After the completion of the primary antibody reaction, it was washed with a TBST solution for 30 minutes, reacted with a secondary antibody, and then treated with an ECL solution in a dark room to sensitize the X-ray film.
실시예Example 1. One. 들깨박Perilla 유래 origin 비검화물Unsaponifiable 내의 비타민 C, Vitamin C within, 피토스테롤Phytosterol , , 폴리코사놀Policosanol 및 스쿠알렌의 함량 측정 And measuring the content of squalene
HPLC(high-performance liquid chromatography) 및 GC(gas chromatography)를 이용하여 들깨박 비검화물 내의 비타민 C, 피토스테롤(phytosterol), 폴리코사놀(policosanol) 및 스쿠알렌(squalene) 함량을 측정한 결과, 들깨박 비검화물의 비타민 E 중에서 가장 풍부한 것은 γ-토코트리에놀(tocotrienol; 330.67mg/100g USM)이었고, 주요 폴리코사놀(policosanol)은 C24(tetracosanol; 857.72mg/100g USM), C26(hexacosanol; 252.88mg/100g USM), C28(octacosanol; 1802.98mg/100g USM), C30(triacontanol; 847.77mg/100g USM)이었으며, 피토스테롤(phytosterol) 중에서 가장 많은 함량을 차지하고 있는 것은 β-시토스테롤(sitosterol; 23016.25mg/100g USM)인 것으로 확인되었다(표 1).As a result of measuring the content of vitamin C, phytosterol, policosanol, and squalene in the unsaponifiable perilla seed using high-performance liquid chromatography (HPLC) and gas chromatography (GC), The most abundant vitamin E was γ-tocotrienol (330.67mg/100g USM), and the main policosanol was C24 (tetracosanol; 857.72mg/100g USM), C26 (hexacosanol; 252.88mg/100g USM), and C28 ( octacosanol; 1802.98mg/100g USM), C30 (triacontanol; 847.77mg/100g USM), and it was confirmed that β-sitosterol (sitosterol; 23016.25mg/100g USM) occupies the most content among phytosterols ( Table 1).
실시예Example 2. 2. 들깨박Perilla 유래 origin 비검화물의Unsaponifiable 세포보호 효과 및 콜라겐 합성 Cell protection effect and collagen synthesis
인간 섬유아세포주인 Hs68 세포에 들깨박 유래 비검화물(이하, PSU)을 처리하고 UVB를 조사하여 세포 생존율 및 세포 내 콜라겐 생성량을 측정하였다.Hs68 cells, a human fibroblast cell line, were treated with an unsaponifiable product (hereinafter, referred to as PSU) derived from perilla and were irradiated with UVB to measure cell viability and intracellular collagen production.
우선, 들깨박 비검화물과 들깨박 메탄올 추출물의 세포보호 효과를 비교한 결과, UVB 조사군(PSU 무처리)이 UVB/PSU 무처리 대조군에 비해 세포 생존율이 약 75% 수준으로 감소하였으나, PSU(1, 2.5 또는 5㎍/㎖) 또는 들깨박 메탄올 추출물(1, 2.5 또는 5㎍/㎖)을 전처리한 후 UVB를 조사하면 농도의존적으로 세포 생존율이 증가하는 것을 확인하였다. 특히 1㎍/㎖ 농도의 PSU 처리군은 UVB 조사군(PSU 무처리)에 비해 세포 생존율이 유의적으로 증가하였으나, 1㎍/㎖ 농도의 들깨박 메탄올 추출물 처리군에서는 유의적 차이가 없음을 확인하였다. 이를 통해, 들깨박 비검화물은 들깨박 메탄올 추출물에 비해 정상 섬유아세포의 증식에서 UVB에 대한 세포보호 효과가 우수하고, 낮은 농도에서도 세포보호 효과가 우수함을 알 수 있었다(도 1A).First, as a result of comparing the cytoprotective effect of the non-saponifiable perilla seed and methanol extract of perilla seed, the UVB irradiation group (no PSU treatment) decreased the cell viability to about 75% compared to the UVB/PSU-free control group, but PSU ( 1, 2.5 or 5 ㎍ / ㎖) or perilla seed meal methanol extract (1, 2.5 or 5 ㎍ / ㎖) was pretreated and irradiated with UVB, it was confirmed that the cell viability increased in a concentration-dependent manner. In particular, the PSU treatment group at a concentration of 1㎍/㎖ significantly increased cell viability compared to the UVB irradiation group (no PSU treatment), but it was confirmed that there was no significant difference in the methanol extract treatment group at the concentration of 1㎍/mL perilla seeds. I did. Through this, it was found that the non-saponifiable perilla perilla extract has excellent cell protection effect against UVB in the proliferation of normal fibroblasts compared to the methanol extract of perilla perilla, and has excellent cell protection effect even at a low concentration (Fig. 1A).
또한, UVB가 조사되지 않은 Hs68 세포에서 PSU 처리군(1, 2.5 또는 5㎍/㎖)과 PSU 무처리 대조군 간의 세포 생존율은 유사한 수준으로 나타나 PSU가 세포 독성이 없음을 알 수 있었다(도 1B). 또한, UVB 조사군(PSU 무처리)이 UVB/PSU 무처리 대조군에 비해 세포 생존율이 약 74% 수준으로 감소하였으나, PSU를 전처리한 후 UVB를 조사하면 PSU 농도의존적으로 세포 생존율이 증가하는 것을 확인하였으며(도 1C), 이를 통해, PSU는 정상 섬유아세포의 증식에서 UVB에 대한 세포보호 효과가 있음을 알 수 있었다.In addition, in Hs68 cells not irradiated with UVB, the cell viability between the PSU-treated group (1, 2.5, or 5 µg/ml) and the PSU-free control group was similar, indicating that PSU had no cytotoxicity (Fig. 1B). . In addition, the UVB irradiation group (no PSU treatment) decreased the cell viability to about 74% level compared to the UVB/PSU untreated control group, but it was confirmed that the cell viability increased depending on the PSU concentration when UVB was irradiated after pretreatment of PSU. (Fig. 1C), through this, it was found that the PSU has a cytoprotective effect against UVB in the proliferation of normal fibroblasts.
또한, UVB가 조사되지 않은 Hs68 세포에 PSU를 농도별로 처리하여 세포 내 콜라겐 생성량을 측정한 결과, PSU 5㎍/㎖ 처리군은 PSU 무처리군에 비해 콜라겐 생성량이 유의적으로 증가한 것을 확인하였고(도 1D), UVB 조사군은 UVB 비조사군에 비해 콜라겐 생성량이 현저한 수준으로 감소하였으나, PSU를 전처리한 후 UVB를 조사하면 PSU 농도의존적으로 콜라겐 생성량이 증가한 것을 확인하였다(도 1E). In addition, as a result of measuring intracellular collagen production by treating PSU at different concentrations in Hs68 cells not irradiated with UVB, it was confirmed that the PSU 5 ㎍ / ㎖ treatment group significantly increased the collagen production amount compared to the PSU untreated group ( 1D), the amount of collagen production in the UVB-irradiated group decreased to a remarkable level compared to the non-UVB-irradiated group, but it was confirmed that the amount of collagen production increased depending on the PSU concentration when UVB was irradiated after pretreatment of the PSU (FIG. 1E ).
실시예Example 3. 3. 들깨박Perilla 유래 origin 비검화물의Unsaponifiable ROSROS 생성량 억제 효과 분석 Analysis of production inhibition effect
들깨박 유래 비검화물(PSU)의 ROS 생성량 억제 효과를 분석한 결과, UVB 조사군(PSU 무처리)은 UVB/PSU 무처리 대조군에 비해 ROS 생성량이 약 2배 이상 증가하였으나, PSU를 전처리한 후 UVB를 조사하면 UVB 조사에 의해 증가된 ROS 생성량이 유의적으로 감소하는 것을 확인하였다(도 2).As a result of analyzing the effect of inhibiting the amount of ROS production of the unsaponifiable (PSU) derived from perilla seeds, the amount of ROS produced in the UVB irradiated group (no PSU treatment) increased by about two times compared to the control group without UVB/PSU, but after pretreatment of the PSU. When irradiated with UVB, it was confirmed that the amount of ROS production increased by UVB irradiation significantly decreased (FIG. 2).
실시예Example 4. 4. 들깨박Perilla 유래 origin 비검화물의Unsaponifiable MMPMMP 단백질 생성 억제 효과 분석 Protein production inhibition effect analysis
MMPs(matrix metalloproteinases) 단백질은 피부의 ECM(extracellular matrix) 구성 단백질을 분해하는 효소로, 노화, UV 또는 열 등의 다양한 스트레스에 의해 각질(keratinocyte)과 진피 섬유아세포(dermal fibroblast)에서 MMP의 생성이 증가하면 ECM의 주성분인 콜라겐과 엘라스틴의 양이 감소되어 피부는 탄력을 잃고 주름이 생기게 된다. 사람의 피부에는 19종의 MMP가 존재하는데 이 중 MMP-1, MMP-3 및 MMP-9만이 자외선 조사에 의하여 발현이 증가한다. 특히, MMP-1은 주요 콜라겐 분해효소로 피부의 주요 콜라겐 형태인 type-Ⅰ과 Ⅲ를 분해시키는데 type-1이 80~90%를 차지하기 때문에 MMP-1의 영향력이 가장 크며, type-1 콜라겐은 진피 섬유아세포에서 콜라겐의 전구체인 프로콜라겐(procollagen)의 형태로 합성 분비된다고 알려져 있다. MMP-1에 의해 분해된 콜라겐은 이어서 MMP-3에 의해 더 분해되며, MMP-3는 pro MMP-1을 활성화시킨다고 알려져 있다. 이에 따라, UVB 자극에 의해 증가된 MMP 단백질에 대한 들깨박 비검화물(PSU)의 영향을 알아보기 위해 세포 내 MMP-1 및 MMP-3의 생성량 및 mRNA 발현량을 측정하였다. MMPs (matrix metalloproteinases) proteins are enzymes that break down the proteins that make up the skin's extracellular matrix (ECM), and the production of MMPs in keratinocytes and dermal fibroblasts is caused by various stresses such as aging, UV or heat. When it increases, the amount of collagen and elastin, which are the main components of ECM, decreases, causing the skin to lose elasticity and wrinkles. There are 19 types of MMPs in human skin, of which only MMP-1, MMP-3 and MMP-9 increase their expression by UV irradiation. In particular, MMP-1 is a major collagen-degrading enzyme that decomposes type-I and Ⅲ, the main collagen types in the skin. Since type-1 accounts for 80-90%, MMP-1 has the greatest influence, and type-1 collagen Is known to be synthesized and secreted in the form of procollagen, a precursor of collagen, from dermal fibroblasts. Collagen degraded by MMP-1 is then further degraded by MMP-3, and MMP-3 is known to activate pro MMP-1. Accordingly, the amount of production of MMP-1 and MMP-3 and the amount of mRNA expression in cells were measured to determine the effect of the non-saponifiable perilla (PSU) on the MMP protein increased by UVB stimulation.
그 결과, UVB 조사군은 UVB/PSU 무처리 대조군에 비해 MMP-1 및 MMP-3의 생성량이 유의적으로 증가하였으나, PSU를 전처리한 후 UVB를 조사하면 UVB 조사군(PSU 무처리)에 비해 MMP-1 및 MMP-3의 생성량이 PSU 농도의존적으로 감소한 것을 확인하였다(도 3A 및 B). 또한, MMP-1 및 MMP-3의 mRNA 발현량 측정 결과도, 상기 MMP-1 및 MMP-3 생성량 결과와 매우 유사하게 나타나는 것으로 확인되었다(도 3C 및 D).As a result, the UVB irradiation group significantly increased the amount of MMP-1 and MMP-3 production compared to the UVB/PSU untreated control group, but when UVB was irradiated after pretreatment of the PSU, compared to the UVB irradiation group (no PSU treatment). It was confirmed that the amount of production of MMP-1 and MMP-3 decreased depending on the PSU concentration (FIGS. 3A and B). In addition, it was confirmed that the measurement results of the mRNA expression levels of MMP-1 and MMP-3 also appear very similar to the results of the production of MMP-1 and MMP-3 (FIGS. 3C and D).
이를 통해, 본 발명의 PSU는 MMP-1 및 MMP-3의 발현과 생성을 모두 저해하여 콜라겐 분해를 억제함으로써 자외선에 의한 피부 광노화를 효과적으로 예방할 수 있을 것으로 사료되었다.Through this, it was thought that the PSU of the present invention could effectively prevent skin photoaging caused by ultraviolet rays by inhibiting collagen degradation by inhibiting both the expression and production of MMP-1 and MMP-3.
실시예Example 5. 5. 들깨박Perilla 유래 origin 비검화물의Unsaponifiable MAPKMAPK 인산화 억제 효과 분석 Analysis of inhibitory effect on phosphorylation
들깨박 유래 비검화물(PSU)이 MAPK(mitogen activated protein kinase)인 ERK, JNK 및 p38의 인산화에 미치는 영향을 분석하였다. JNK 및 ERK 인산화는 인산화된 MAPK 발현량/MAPK 발현량으로 나타내었다.The effect of unsaponifiable (PSU) derived from perilla seeds on the phosphorylation of ERK, JNK and p38, which are mitogen activated protein kinase (MAPK), was analyzed. JNK and ERK phosphorylation were expressed as phosphorylated MAPK expression/MAPK expression.
그 결과, JNK 인산화 수준은 UVB/PSU 무처리 대조군에 비해 UVB 조사군(PSU 무처리)에서 현저하게 증가하였으나, PSU를 농도별(1, 2.5 또는 5㎍/㎖)로 전처리한 후 UVB를 조사하면 UVB 조사군(PSU 무처리)에 비해 JNK의 인산화 수준이 PSU 농도의존적으로 61%, 30.9%, 19.7%까지 감소하는 것을 확인하였다(도 4A).As a result, the level of JNK phosphorylation was significantly increased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group, but UVB irradiation after pretreatment of PSU at each concentration (1, 2.5 or 5㎍/㎖) When compared to the UVB irradiation group (no PSU treatment), it was confirmed that the phosphorylation level of JNK decreased to 61%, 30.9%, and 19.7% depending on the PSU concentration (FIG. 4A).
또한, ERK 및 p-38 인산화 수준도 상기 JNK 인산화 수준 측정 결과와 매우 유사하게 나타났으며, PSU를 농도별로 전처리한 후 UVB를 조사하면 ERK 인산화 수준의 경우 PSU 농도의존적으로 78.9%, 45.3%, 12.8%까지 감소하고(도 4B), p-38 인산화 수준의 경우 PSU 농도의존적으로 60.9%, 27.8%, 15.3%까지 감소하는 것을 확인하였다(도 4C). 이를 통해, 본 발명의 PSU가 MAPK의 인산화를 억제하여 콜라겐 분해효소의 발현을 억제할 수 있음을 알 수 있었다.In addition, ERK and p-38 phosphorylation levels were also very similar to those of the JNK phosphorylation level measurement results, and when UVB irradiation after pretreatment of PSU concentrations, ERK phosphorylation levels were 78.9%, 45.3%, depending on PSU concentration, It was confirmed that it decreased to 12.8% (FIG. 4B), and the p-38 phosphorylation level decreased to 60.9%, 27.8%, and 15.3% depending on the PSU concentration (FIG. 4C). Through this, it was found that the PSU of the present invention can inhibit the expression of collagen-degrading enzyme by inhibiting the phosphorylation of MAPK.
실시예Example 6. 6. 들깨박Perilla 유래 origin 비검화물의Unsaponifiable AP-1 전사인자 활성 억제 효과 분석 Analysis of AP-1 transcription factor activity inhibition effect
전사인자인 AP-1은 c-jun과 c-fos 단백질로 구성되어 있으며 UVB 조사에 의해 활성화되며, 활성화된 AP-1 의해 MMPs 생성량도 증가한다. 상기 실험에서 들깨박 유래 비검화물(PSU)이 MMP 단백질의 발현과 생성을 모두 감소시키는 것을 확인하였으며, PSU가 MMP 단백질의 상위조절자인 AP-1의 활성도 감소시키는지 조사하였다. AP-1 활성은 p-c-jun 또는 p-c-fos 발현량/c-jun 또는 c-fos 발현량으로 나타내었다.AP-1, a transcription factor, is composed of c-jun and c-fos proteins and is activated by UVB irradiation, and the amount of MMPs produced is also increased by activated AP-1. In the above experiment, it was confirmed that the unsaponifiable (PSU) derived from perilla seeds reduced both the expression and production of the MMP protein, and it was investigated whether PSU also reduced the activity of AP-1, the upper regulator of the MMP protein. AP-1 activity was expressed as the expression level of p-c-jun or p-c-fos/c-jun or c-fos expression.
그 결과, c-fos 인산화 수준은 UVB/PSU 무처리 대조군에 비해 UVB 조사군(PSU 무처리)에서 현저하게 증가하였으나, PSU를 농도별(1, 2.5 또는 5㎍/㎖)로 전처리한 후 UVB를 조사하면 c-fos 인산화 수준이 PSU 농도의존적으로 68.1%, 40.1%, 23.3%까지 감소하는 것을 확인하였다(도 5A).As a result, the c-fos phosphorylation level was significantly increased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group, but after pretreatment of PSU at each concentration (1, 2.5 or 5㎍/㎖) When investigated, it was confirmed that the c-fos phosphorylation level decreased to 68.1%, 40.1%, and 23.3% depending on the PSU concentration (FIG. 5A).
또한, c-jun 인산화 수준도 상기 c-fos 인산화 수준 측정 결과와 매우 유사하게 나타났으며, PSU를 농도별로 전처리한 후 UVB를 조사하면 c-jun 인산화 수준이 PSU 농도의존적으로 88.6%, 80.0%, 63.8%까지 감소하는 것을 확인하였다(도 5B). 이를 통해, 본 발명의 PSU가 AP-1 전사인자의 인산화를 억제하여 콜라겐 분해효소의 발현을 억제할 수 있음을 알 수 있었다.In addition, the c-jun phosphorylation level was also very similar to the c-fos phosphorylation level measurement result, and when UVB was irradiated after pretreatment of the PSU by concentration, the c-jun phosphorylation level was 88.6%, 80.0% depending on the PSU concentration. , It was confirmed that it decreased to 63.8% (FIG. 5B). Through this, it was found that the PSU of the present invention can inhibit the expression of collagen degrading enzyme by inhibiting the phosphorylation of AP-1 transcription factor.
실시예Example 7. 7. 들깨박Perilla 유래 origin 비검화물의Unsaponifiable TGFTGF -β 경로 활성 증진 효과 분석-β pathway activity enhancement effect analysis
피부의 세포외기질은 노화되는 동안 점차적으로 분열되며, 노화된 인간 피부에서 진피의 세포외기질과 관련된 유전자들은 TGF-β 경로를 통해 감소된다. TGF-β는 Smad2/3의 인산화를 촉진시키고 Smad7의 경우 활성화된 TGFR1에 결합하기 위해 Smad2와 Smad3과 경쟁적으로 작용한다. UVB에 의해 증가된 ROS는 진피 섬유아세포 내에서 TGF-β의 작용을 저해하고 세포 내 전사인자인 AP-1을 활성화시킨다고 알려져 있다. 이에 따라, Type 1 procollagen 합성의 주요 조절인자인 TGFβ/smad 신호전달경로에 들깨박 유래 비검화물(PSU)이 미치는 영향에 대해 분석하였다.The extracellular matrix of the skin is gradually divided during aging, and genes related to the extracellular matrix of the dermis in aged human skin are reduced through the TGF-β pathway. TGF-β promotes phosphorylation of Smad2/3 and, in the case of Smad7, competes with Smad2 and Smad3 to bind to activated TGFR1. It is known that ROS increased by UVB inhibits the action of TGF-β in dermal fibroblasts and activates AP-1, an intracellular transcription factor. Accordingly, the effect of perilla seed-derived unsaponifiable (PSU) on the TGFβ/smad signaling pathway, a major regulator of
그 결과, TGF-β1 및 Smad2/3의 발현량은 UVB/PSU 무처리 대조군에 비해 UVB 조사군(PSU 무처리)에서 현저하게 감소하였으나, PSU를 농도별(1, 2.5 또는 5㎍/㎖)로 전처리한 후 UVB를 조사하면 UVB 조사군(PSU 무처리)에 비해 TGF-β1과 Smad2/3의 발현량이 PSU 농도의존적으로 증가하는 것을 확인하였다(도 6A 및 B)As a result, the expression levels of TGF-β1 and Smad2/3 significantly decreased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group, but the PSU concentration by concentration (1, 2.5 or 5㎍/㎖) After pretreatment with UVB, it was confirmed that the expression levels of TGF-β1 and Smad2/3 increased in a PSU concentration-dependent manner compared to the UVB irradiation group (no PSU treatment) (Figs. 6A and B).
반면, TGF-β1 및 Smad2/3의 활성을 저해는 Smad7의 발현량은 UVB/PSU 무처리 대조군에 비해 UVB 조사군(PSU 무처리)에서 현저하게 증가하였으나, PSU를 농도별로 전처리한 후 UVB를 조사하면 UVB 조사군(PSU 무처리)에 비해 Smad7의 발현량이 PSU 농도의존적으로 감소한 것을 확인하였다(도 6C).On the other hand, the expression level of Smad7, which inhibits the activity of TGF-β1 and Smad2/3, was significantly increased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group, but UVB was removed after pretreatment of PSU by concentration. When irradiated, it was confirmed that the amount of expression of Smad7 decreased in a PSU concentration-dependent manner compared to the UVB irradiation group (no PSU treatment) (FIG. 6C).
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