유태현 김민경 2019--2 28315 https://aurora.ajou.ac.kr/handle/2018.oak/15211 학위논문(박사)--아주대학교 일반대학원 :응용생명공학과,2019. 2 화장품 산업이 발달함에 따라 보다 기능적이고, 자연주의적인 제품을 선호하는 소비자들의 요구가 급증하면서, 다양한 식물체로부터 인체에 무해하면서 피부 생리 활성에 좋은 화장품 소재 개발의 필요성이 중요해지고 있다. 첫번째로, 숙근 천인국 꽃 추출물의 항산화 효과 및 미백 효능을 관찰하였다. 항산화 효능 시험 결과, 숙근 천인국 꽃 추출물이 높은 페놀함량과 자유라디칼, 양이온 라디칼을 소거하는 기능 및 우수한 항산화 효과를 나타냈다. 쥐의 흑색종 세포(B16F10)와 인체유래 멜라닌 세포를 이용하여 숙근 천인국 꽃 추출물의 미백효능을 확인하였다. 세포 독성이 없는 농도에서 멜라닌이 생성된 양을 관찰한 결과, 두 세포 모두 멜라닌 생성량을 감소시키는 결과를 보였다. 멜라닌 합성에 중요한 역할을 하는 티로시나아제 활성 시험, 티로시나아제 자이모그래피, 루시퍼라아제 활성 시험에서도 두 세포 모두 숙근 천인국 꽃 추출물이 농도 의존적으로 세포 내 티로시나아제 활성 억제 효능을 나타내는 것을 관찰하였다. 웨스턴 블롯과 실시간 중합효소연쇄반응 시험을 진행한 결과, 멜라닌 색소형성에 관여하는 TYR, TRP-1, DCT, MITF의 단백질 및 유전자 발현량이 농도 의존적으로 감소하였다. 게다가, 인체피부와 가장 유사한 시스템인 3D 피부 조직을 이용하여 미백 효능을 확인한 결과 숙근 천인국 꽃 추출물이 양성대조군으로 사용한 코직산보다 훨씬 더 낮은 농도에서 멜라닌 생성 저해 효능이 나타나는 것을 확인하였다. 그리고, 3D 피부 조직에서 줄기세포인자를 처리하여 피부 밝기 지수 비교시험을 진행하였을 때 줄기세포인자를 처리 후 숙근 천인국 꽃 추출물을 처리 시 피부 밝기 지수가 더 높게 나타났다. 마지막으로 인체 일차피부자극 시험을 진행하여 숙근 천인국 꽃 추출물이 피부에 자극이 없는 안전한 소재임을 확인하였고, 숙근 천인국 꽃 추출물이 포함된 로션 제형에 대한 인공 색소침착법을 이용한 미백효과 평가시험을 통해 피부 미백 효능을 재검증하였다. 두번째 연구는 엉겅퀴꽃 추출물의 멜라닌 합성을 증가시키는 효과를 관찰하기 위해서 백인에서 유래된 밝은 색을 나타내는 멜라닌 세포와 아시아인에서 유래된 멜라닌 세포를 사용하여 실험을 진행하였다. 엉겅퀴꽃 추출물은 두 세포 모두 낮은 농도에서 멜라닌 합성과 티로시나아제 활성을 농도 의존적으로 증가시켰다. 페오멜라닌과 유멜라닌의 주요한 멜라닌 표지 인자를 관찰하기 위해서 엉겅퀴꽃 추출물을 두 세포에 처리하여 배양하였고, 과산화수소 산화반응을 시켜 멜라닌을 분해시킨 후에 HPLC로 측정하였다. 엉겅퀴꽃 추출물의 멜라닌 합성 기전은 웨스턴 블롯과 사이클릭 AMP 면역측정법을 통해서 관찰되었고, 엉겅퀴꽃 추출물이 사이클릭 AMP 신호 전달체계를 통한MITF와 티로시나아제를 상향조절시키는 것을 확인하였다. 게다가, 엉겅퀴꽃 추출물은 각질세포와 멜라닌세포가 포함된 3D 피부 조직에서 멜라닌 함량을 상당히 증가시켰다. 또한, 인체 모낭세포 조직에서도 엉겅퀴꽃 추출물의 멜라닌 합성 효능을 관찰하였다. 31명의 피험자를 대상으로 진행한 안전성 시험에서 엉겅퀴꽃 추출물은 어떠한 피부 자극도 나타나지 않았다. 세번째로, 동백꽃 추출물의 항노화 효과 및 각질형성세포와 섬유아세포, 그리고 피부 조직을 이용하여 도시의 대기 오염물질에 의해 유도되는 피부노화를 보호하는 효과를 알아보기 위한 실험을 진행하였다. 동백꽃 추출물이 노화와 연관된 최종당화산물 생성 억제 효능과 콜라겐 사이의 교차결합 저해 및 절단 효능이 있음을 확인하였고, 임상 시험을 진행하여 피부 결, 탄력, 수분 및 진피 치밀도 개선 효능을 검증하였다. 높은 페놀 농도를 갖는 동백꽃 추출물은 항산화 시험을 통해서 DPPH 라디칼과 ABTS+ 의 소거 능력이 농도 의존적으로 나타나는 결과를 보여주었다. 그리고, 동백꽃 추출물이 대기 오염물질에 유도된 세포 내 활성산소종과 MMP-1, AhR 전사 인자에 관여하는 XRE 루시퍼라아제 활성을 저해하는 것을 각질형성세포와 섬유아세포를 통해서 관찰할 수 있었다. 또한, 인체 피부조직 모델에서 동백꽃 추출물이 오염물질에 유도된 지질 과산화 표지로 알려진 MDA의 양을 감소시키고 증가한 MMP-1의 발현을 저해하였으며, 콜라겐 합성도 증가시킴에 따라 동백꽃 추출물이 오염물질에 의해서 손상된 피부를 보호하는 효과가 뛰어남을 증명하였다. 게다가, 동백꽃 추출물이 오염물질에 의해서 탈락된 진피와 표피의 연결 접합부인 DEJ와 주로 세포자멸 작용에서 발생하는 농축핵을 가진 세포 수를 감소시키는 것을 관찰할 수 있었다. 마지막으로 안전성 시험을 통해서 동백꽃 추출물이 피부에 안전한 천연 물질임을 확인하였다. 따라서, 이번 연구는 인체에 무해한 미백, 흑화, 항노화 및 대기오염물질로부터 손상된 피부를 보호하는 효능이 뛰어난 천연 화장품 소재들을 개발하였음을 시사한다. Chapter 1. Introduction ............................................................................................ 1 1.1 Human Skin ......................................................................................................... 1 1.2 Skin Pigmentation ............................................................................................... 2 1.3 Skin Aging ............................................................................................................ 6 1.4 Air pollutants-induced Skin Aging .................................................................... 8 1.5 Cosmetic Ingredients ........................................................................................ 12 1.6 Purpose of the Study ......................................................................................... 14 1.7 References .......................................................................................................... 17 Chapter 2. Inhibition of melanogenesis by Gaillardia aristata flower extract 2.1 Introduction ....................................................................................................... 22 2.2 Materials and Methods ..................................................................................... 24 2.2.1 Chemicals and Reagents ............................................................................... 24 2.2.2 Preparation of GAE ...................................................................................... 24 2.2.3 Antioxidant assay ......................................................................................... 25 2.2.3.1 DPPH Scavenging Activity Assay ......................................................... 25 2.2.3.2 ABTS+ Scavenging Capacity Assay ...................................................... 25 2.2.3.3 Determination of Reducing Capacity ..................................................... 26 2.2.3.4 Determination of Total Phenolic Content ............................................... 26 2.2.4 Cell Culture .................................................................................................. 26 2.2.5 Cell Viability Assay ...................................................................................... 27 2.2.6 Determination of Melanin Content ............................................................... 27 2.2.7 Measurement of Cellular Tyrosinase Activity .............................................. 28 2.2.8 Tyrosinase Zymography ............................................................................... 28 2.2.9 Tyrosinase Luciferase Reporter Assay .......................................................... 29 2.2.10 Western Blotting ......................................................................................... 29 2.2.11 Quantitative Real-Time PCR Analysis ....................................................... 29 2.2.12 Histochemistry of Reconstructed Epidermis .............................................. 30 2.2.13 Evaluation of Inhibitory Efficacy of GAE on the 3D-Human Skin Model ...30 2.2.14 Human Skin Primary Irritation Test ............................................................ 31 2.2.15 Clinical Study ............................................................................................. 32 2.3 Results ................................................................................................................ 34 2.3.1 Antioxidant capacities of GAE ..................................................................... 34 2.3.2 Effect of GAE on Melanin Production in HEMa-DP Cells .......................... 36 2.3.3 Effect of GAE on Cellular Tyroisnase Inhibition in HEMa-DP Cells .......... 37 2.3.4 Evaluation of Antimelanogenesis Activity in B16F10 Cells ........................ 39 2.3.5 Effect of GAE on Melanogenesis-Related Proteins and Gene Expression ... 41 2.3.6 Effect of GAE in Human Epidermal Equivalents ......................................... 43 2.3.7 Effect of GAE on SCF-stimulated Pigmentation in Human Epidermal Equivalents ..................................................................................................... 44 2.3.8 Human Skin Primary Irritation Test of GAE ................................................ 46 2.3.9 Clinical Study for the Evaluation of the Whitening Effects .......................... 47 2.4 Discussion .......................................................................................................... 53 2.5 References .......................................................................................................... 55 Chapter 3. The hyper-pigmentation effect of Cirsium japonicum flower extract in the regulation of melanogenesis 3.1 Introduction ....................................................................................................... 59 3.2 Materials and Methods ..................................................................................... 61 3.2.1 Materials ....................................................................................................... 61 3.2.2 Extraction of Cirsium japonicum Flower ...................................................... 61 3.2.3 Cell Culture and Viability ............................................................................. 62 3.2.4 Melanin Content and Cellular Tyrosinase Activity ....................................... 62 3.2.5 Cellular tyrosinase activity ........................................................................... 62 3.2.6 cAMP AMP Immnuassay ............................................................................. 63 3.2.7 Western blot Analysis ................................................................................... 63 3.2.8 3D Skin Model .............................................................................................. 64 3.2.9 Alkaline H2O2 Oxidation .............................................................................. 64 3.2.10 HPLC Analysis ........................................................................................... 65 3.2.11 Ex-vivo Human Hair Follicles ..................................................................... 65 3.2.12 Skin Irritation Test on Human Skin ............................................................ 66 3.2.13 Statistical Analysis ..................................................................................... 67 3.3 Results ................................................................................................................ 67 3.3.1 Effect of C. japonicum Flower Extract (CFE) on Cell Viability and Melanin Content in Light and Moderate Pigmented Human Melanocytes ................... 67 3.3.2 Analysis on Melanin Markers by Process of Alkaline H2O2 Oxidation ........ 68 3.3.3 Effect of CFE on Cellular Tyrosinase Activity ............................................. 70 3.3.4 Effect of CFE by Activation on Cyclic AMP Signaling Pathway ................. 71 3.3.5 Melanogenic Effect of CFE on 3D Skin Model ............................................ 73 3.3.6 Effect on Pigmentation on Ex vivo Human Hair Follicles with CFE ............ 74 3.3.7 Evaluation of Safety Test .............................................................................. 75 3.4 Discussion .......................................................................................................... 76 3.5 References .......................................................................................................... 78 Chapter 4. Protective effects of Camellia japonica flower extract against urban air pollutants 4.1 Introduction ....................................................................................................... 81 4.2 Materials and Methods ..................................................................................... 83 4.2.1 Materials ....................................................................................................... 83 4.2.2 Extraction of Camellia japonica Flower ....................................................... 83 4.2.3 Preparation of Urban dust ............................................................................. 84 4.2.4 Cell Culture .................................................................................................. 84 4.2.5 Cell Viability ................................................................................................ 84 4.2.6 Antioxidant Assay ........................................................................................ 85 4.2.6.1 DPPH Assay .......................................................................................... 85 4.2.6.2 ABTS Radical Scavenging Assay........................................................... 85 4.2.6.3 Total Phenolic Content ........................................................................... 85 4.2.6.4 Total Flavonoid Content ........................................................................ 86 4.2.7 Intracellular ROS Production ....................................................................... 86 4.2.8 XRE Luciferase Activity .............................................................................. 86 4.2.9 MMP-1 Inhibition ........................................................................................ 87 4.2.10 Real-time PCR ............................................................................................ 87 4.2.11 Microarray in Fibroblasts ........................................................................... 87 4.2.12 Anti-glycation Assay .................................................................................. 88 4.2.13 Inhibition of Collagen-AGEs Crosslinking Assay ...................................... 88 4.2.14 Breaking of Collagen-AGEs Crosslinking Assay ....................................... 89 4.2.15 Anti-aging on Human Living Skin Explants ............................................... 90 4.2.16 Safety Test .................................................................................................. 91 4.2.16.1 Photo-toxicity Test ............................................................................... 91 4.2.16.2 HET-CAM Test .................................................................................... 91 4.2.16.3 Skin Cumulative Irritation Test on Human Skin .................................. 94 4.2.17 Clinical Study ............................................................................................. 95 4.2.18 Statistical Analysis ..................................................................................... 98 4.3 Results ................................................................................................................ 99 4.3.1 Free Radical Scavenging Activity, Total Phenolic and Flavonoid Content of CJFE ............................................................................................................... 99 4.3.2 Anti-aging activity of CJFE ........................................................................ 101 4.3.3 A Clinical Study of Anti-aging Effect on Human skin ................................ 104 4.3.4 Inhibitory Effect on CJFE on Urban Pollutants Induced ROS Production .. 115 4.3.5 Inhibitory effect of CJFE on urban pollutants induced AhR-signaling pathway ........................................................................................................ 118 4.3.6 Inhibitory Effect of CJFE on Urban Pollutants Induced MMP-1 ................ 120 4.3.7 Expression of Genes Involved in Fibroblasts by Microarray analysis ........ 121 4.3.8 Protective effect of CJFE on Human living skin Explants .......................... 123 4.3.9 Evaluation of Safety Test ............................................................................ 126 4.3.9.1 The Clinical Safety Test on Human skin .............................................. 126 4.3.9.2 The Eye Irritation Potential Evaluation Test ........................................ 127 4.3.9.3 The Photo-toxicity Evaluation Test ...................................................... 130 4.4 Discussion ........................................................................................................ 131 4.5 References ........................................................................................................ 133 Chapter 5. Conclusion .......................................................................................... 139 Summary in Korean .............................................................................................. 143 eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. Studies on the biological activities of natural flower extracts for cosmetic applications Kim, Minkyung Thesis 아주대학교 일반대학원 Kim, Minkyung 일반대학원 응용생명공학과 2019. 2 Doctoral I804:41038-000000028315 http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000028315 The demand of consumers who prefer more functional and natural products has increased by the development of the cosmetics industry. Accordingly, it is important to develop harmless cosmetic ingredients with effective biological activity in skin. The purpose of the first study was to determine the anti-melanogenic and anti-oxidant properties of Gaillardia aristata flower extract (GAE). GAE possessed anti-oxidant characteristics as free radical-scavenging capacity and reducing power. Melanogenesis inhibition by GAE was investigated in cultivated cells and in a human skin model. In cultivated cells, the melanogenesis regulatory effect of GAE was evaluated using melanin content, intracellular tyrosinase activity. In addition, the expression of melanogenesis-related proteins was determined by western blot assay and real-time PCR. GAE reduced the amount of melanin in B16F10 and normal human epidermal melanocyte cells and suppressed intracellular tyrosinase activity in a dose-dependent pattern. Also, GAE significantly decreased the expression of melanogenesis-related proteins (microphthalmia associated transcription factor, tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase). Real-time PCR results revealed a down-regulation of the mRNAs of these proteins. In the three-dimensional human skin model, GAE showed the inhibitory effect on melanin formation at a much lower concentration than kojic acid used as a positive control. GAE significantly increased the skin lightening in 3D skin tissue hyper-pigmented by stem cell factor (SCF). In addition, the safety of GAE on human skin was confirmed by skin primary irritation test. Visual assessment, melanin index and skin brightness (L* value) were improved by the formulation containing 0.05% GAE on clinical study for whitening effect. These results indicate the potential of GAE for use in suppressing skin pigmentation. The purpose of the second study was to investigate the melanogenic effect on Cirisum japonicum flower extract (CFE) in light and moderate pigmented human melanocytes. CFE increased the melanin content in both cells in a dose-dependent manner. To confirm major melanin markers of pheomelanin and eumelanin in both cells cultured with treatment of CFE, high-performance liquid chromatography (HPLC) analysis was performed by alkaline H2O2 Oxidation. CFE significantly increased the cellular tyrosinase activity at low concentration. The mechanism on pigmentation effect of CFE was demonstrated via cyclic AMP immunoassay and western blot assay. CFE increased the melanin synthesis by up-regulating MITF and tyrosinase expression via cAMP signaling pathway. Furthermore, CFE considerably increased the melanin content on a reconstituted 3-dimensional human skin containing normal epidermal keratinocytes and melanocytes. The potential pro-pigmenting effect of CFE on ex vivo human hair follicles was additionally evaluated by Fontana Masson staining. CFE showed a substantial increase of melanin in the hair bulb and shaft on human hair follicles. No irritation reaction was occurred on human skin with treatment of CFE. Consequently, C. japonicum flower extract with the hyperpigmentation effect suggests that it can be variously applied to potential agent as melanogenesis stimulator on human skin and hair. The third study was investigated anti-aging and protective effects of Camellia japonica flower extract (CJFE) against urban air pollutants using in vitro and ex vivo model. CJFE showed anti-aging activity such as skin texture, elasticity, hydration and dermal density on human skin plus the effect of anti-glycation and the effect of inhibition and breaking on collagen-AGEs crosslinking in vitro. CJFE with high phenolic concentration showed the anti-oxidative activity of CJFE on scavenging capacity of 1,2-diphenyl-2-picrylhydrazyl (DPPH) radicals and 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS+) in a concentration dependent manner. CJFE inhibited urban air pollutant-induced ROS generation, MMP-1 production and a XRE-luciferase activity indicating the AhR transactivation. In addition, CJFE showed an excellent protective activity against pollutant-induced deteriorating effect in ex vivo model. CJFE reduced the level of pollutant-induced malondialdehyde (MDA), lipid peroxidation marker, inhibited MMP-1 expression and increased collagen synthesis. It also reduced the cell numbers with pyknotic nuclei (mainly occurring in apoptosis) and detachment of dermo-epidermal junction (DEJ) induced by pollutants. Finally, the safety test revealed that CJFE is a safe natural substance on skin. It is proposed that CJFE can be used as a safe material with anti-aging and protective effects against pollutant-induced skin damages. Thus, these studies suggest that harmless natural cosmetic materials have been developed, which are highly effective of whitening, melanogenic, anti-aging and anti-pollution.