The study on verification of protection mechanisms of skin aging caused by UVB and major compounds purification from fermentation by Lactovacillus casei in chestnut(Castanea crenata) bur : 발효 밤송이 추출물의 유효성분 분석 및 UVB에 의한 피부노화 방어 메커니즘 검증

다국어 초록 (Multilingual Abstract)

Skin aging is divided into the intrinsic aging occurring due to individual genes as each individual is getting older & the photoaging caused by oxidative stress due to substances from outside & UV. Of the two, the photoaging has clinical characteristics; wrinkles on skin are early formed due to UV, skin gets dry, skin pigmentation is caused, and peripheral blood vessels are extended. Also atypical cell, decrease of collagenic, and disappearance of elastic fibers are shown in the histological characteristics. Skin aging is caused by accumulation of pro-oxidants, by generating reactive oxygen species, ROS & free radical in our body with constant stimuli from outside. Therefore antioxidants can make human aging be delayed in a wide sense.
This study aimed to separate excellent active ingredients from checking pharmaceutic active of cosmetics after a chestnut burr as by-product of chestnut fruit tree was fermented, and to identify correlations among the ingredients through animal testing. Also it's prescribed so that the active ingredients of cosmetics could be stably collected into cosmetics for industrial application of these correlations .
To evaluate antioxidant active, it's tested by ABTS radical scavenging, Electron donating ability (EDA), Hydrogen peroxide (H2O2) scavenging, Superoxide dismutase (SOD)-like, Nitrite-scavenging, Superoxide anion radical (O2-) scavenging, Reducing Power measurement method, and Collagenase inhibition, Elastase inhibition was measured to assess ability to improve wrinkles. As a result of testing, it's shown to be the most excellent active among groups fermented for 6 days after strain 2×106 cfu/ml was inoculated, and then the active, which was tested and segmented by a solvent, was identified to be in ethyl acetate layer. On basis of this, the active was separated and purified, and 4 compounds of major substances such as ellagic acid, 1-O-Galloyl-â-D-glucose, Quercetin 3-O-â-D-glucopyranoside, (+)-Catechin were identified. Among them, as a result of analyzing protein & DNA revealed in relation to wrinkles by UVB of ellagic acid through test of western blot & RT-PCR, phosphorylation of PKCá, ERK, P38, C-JUN protein & revelation of MMPs protein were significantly obstructed, and revelation ratios of MMP-1, 9 DNA were respectively reduced. Also as a result of measuring changes of skin thickness in groups appled by ellagic acid through animal test using Hairless mice, groups treated with 50mg/ml of concentration were reduced to 52% and were shown to be even stronger stainability in the result of checking collagen fibers stained by Masson’s trichrome, compared with control groups.
As a result of stability of liquid crystal containing ellagic acid, changes of temperature, pH, viscosity were stabilized and cosmetics(toner, essence) with them was also stabilized. As a result of testing with patch for stability, all was observed to be safe. On the basis of these results, extracts of fermentation in chestnut bur are considered to be available for developing materials of cosmetics with improvement effect on wrinkles.

국문 초록 (Abstract)

피부의 노화는 나이가 증가하면서 개인적인 유전자에 의하여 발생하는 내재적 노화(intrinsic aging)와 자외선과 외부 물질에 의한 산화적 스트레스에 의한 광노화(photoaging)로 분류한다. 이중 광노화는 자외선(ultraviolet, UV)에 의하여 피부 주름이 조기에 형성되고 피부가 건조해지며, 색소가 침착되고, 말초혈관이 확장되는 임상적인 특징을 나타낸다. 또한 세포의 비전형화(atypical cell), 아교질의 감소, 탄력섬유가 소실되는 조직학적 특징을 나타낸다. 우리 인체는 외부로부터 끊임없는 자극을 받아 활성산소 (reactive oxygen species, ROS)와 유리기를 생성함으로써, 산화물질 (pro-oxidants)을 축적하여 노화가 일어난다. 그러므로 노화 방지제는 넓은 의미에서 인간의 노화를 지연시킬 수 있다.
이 연구의 목적은 밤나무 과수의 부산물인 밤송이를 발효하여 화장품약리활성을 확인하여 우수한 유효성분을 분리하고, 이를 동물실험을 통해 상관관계를 확인하였다. 또한 이의 산업적 응용을 위해 화장품 활성성분이 화장품에 안정적으로 포집될 수 있도록 처방하였다.
항산화 활성을 평가하기 위하여 ABTS radical scavenging, Electron donating ability (EDA), Hydrogen peroxide (H2O2) scavenging, Superoxide dismutase (SOD)-like, Nitrite-scavenging, Superoxide anion radical (O2-) scavenging, Reducing Power측정 방법으로 실험하였으며, 주름개선능을 평가하기 위하여 Collagenase inhibition, Elastase inhibition을 측정 하였다. 한 결과 균주 2×106 cfu/ml 접종 후 6일간 발효한 군에서 가장 우수한 활성을 나타내었으며, 이를 용매 분획하여 실험하여 에틸아세테이트층이 가장 우수한 활성을 확인하였다. 이를 바탕으로 분리 정제하여 주요물질인 ellagic acid, 1-O-Galloyl-â-D-glucose, Quercetin 3-O-â-D-glucopyranoside, (+)-Catechin 4종의 화합물을 확인하였다. 이중 ellagic acid의 UVB에 의한 주름관련 protein과 DNA발현을 western blot과 RT-PCR실험을 통해 분석한 결과 PKCá, ERK, P38, C-JUN protein의 인산화와 MMPs protein의 발현을 유의적으로 저해시켰으며, MMP-1, 9 DNA의 발현량을 감소시켰다. 또한 Hairless mice를 이용한 동물실험을 통해 ellagic acid를 도포한 군에서 표피 두께변화를 측정한 결과 50mg/ml의 농도를 처리군이 대조군에 비해 52% 감소하였으며, Masson’s trichrome염색에 의한 아교섬유의 염색을 확인한 결과에서도 50mg/ml의 농도를 처리군이 대조군에 비해 더욱 강한 염색성을 나타내었다.
ellagic acid를 함유한 liquid crystal의 안정성 결과 temperature 변화, pH, viscosity가 안정하였으며 이를 함유한 화장품(toner, essence)역시 안정하였다. 안전성 실험으로 패치 테스트 결과 모두 안전한 것으로 관찰되었다. 이러한 실험결과 밤송이 발효 추출물은 주름개선 효과를 가진 화장품 원료 개발에 이용이 가능할 것으로 사료된다.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 10
• 1. Materials 10
• 1) Chemicals and Instruments 10
• 2) Plant Materials 11
• 3) Fermentation 12
• (1) Culture and inoculation 12
• (2) Establishment of the optimized extraction conditions 12
• (3) Extract 14
• (4) pH measurement 14
• 4) Isolation 17
• 5) Instrumentation 18
• 2. Methods 19
• 1) Anti-oxidative effect 19
• (1) ABTS radical scavenging assay 19
• (2) Electron donating ability (EDA) assay 19
• (3) Hydrogen peroxide (H2O2) scavenging activity 20
• (4) Superoxide dismutase (SOD)-like activity 20
• (5) Nitrite-scavenging assay 20
• (6) Superoxide anion radical (O2-) scavenging assay 21
• (7) Reducing Power 21
• 2) Anti-Wrinkle effect 22
• (1) Collagenase inhibition activity 22
• (2) Elastase inhibition activity 22
• 3) Anti-microbial activity 23
• (1) Growth of microorganisms 23
• (2) Inhibitory effect by the diffusion method 23
• 4) Determination of total phenolics and flavonoids contents 24
• 5) Inhibitory effect of ethanol extracts of fermentation by 25
• (1) Cell viability assay 25
• (2) UVB irradiation on CCRF S-180 II cells 26
• (3) Determination of intercellular reactive oxygen species 26
• (4) DNA fragmentation patterns in CCRF S-180 II cells of 27
• (5) Protein analysis with western blot 28
• (6) Real time PCR 29
• (7) Reverse transcription(RT)-PCR 29
• 6) Experimental animals and skin administration 30
• (1) Histological examination 30
• 7) Reparation of liquid crystal 31
• (1) Microscopy analysis 32
• (2) X-ray diffraction (XRD) 32
• (3) Transmission scanning microscopy (TEM) 33
• 8) Preparation of liquid crystal containing cosmetic products 34
• (1) Preparation of cosmetic products 34
• (2) Particle size and size distribution 38
• 9) Stability and safety test of cosmetic product 39
• (1) Stability test 39
• (2) Safety test 41
• 10) Statistical analysis 42
• Ⅲ. Results 43
• 1. Yield 43
• 2. Result of anti-oxidation effect 44
• 1) ABTS radical scavenging assay 44
• 2) Electron donating ability (EDA) assay 46
• 3) Hydrogen peroxide (H2O2) scavenging activity 48
• 4) Superoxide dismutase (SOD)-like activity 50
• 5) Nitrite-scavenging assay 52
• 6) Superoxide anion radical (O2-) scavenging assay 54
• 7) Reducing Power 56
• 3. Result of anti-wrinkle effect 58
• 1) Collagenase inhibition activity 58
• 2) Elastase inhibition activity 60
• 4. Result of anti-microbial activity 62
• 1) Measurement of growth inhibition zone 62
• 5. Result of anti-oxidation effect of fractions isolated from CBh 65
• 1) ABTS+ radical scavenging assay 65
• 2) Electron donating ability (EDA) assay 66
• 3) Hydrogen peroxide (H2O2) scavenging activity 67
• 4) Superoxide dismutase (SOD)-like assay 68
• 5) Nitrite-scavenging ability 69
• 6) Superoxide anion radical (O2-) scavenging assay 70
• 7) Reducing power 71
• 6. Result of Anti-wrinkle Effect of fractions isolated from CBh 72
• 1) Collagenase inhibition activity 72
• 2) Elastase inhibition activity 74
• 7. Result of anti-microbial activity of fractions isolated from CBh 75
• 1) Measurement of growth inhibition zone 75
• 8. Determination of total phenolics and flavonoids contents 79
• 9. Identification of compounds isolated of CBhE 80
• 1) Identification of compound 1 80
• 2) Identification of compound 2 88
• 3) Identification of compound 3 96
• 4) Identification of compound 4 102
• 10. Inhibitory effect of ethanol extracts of fermentation by Lactobacillus 110
• 1) Cell viability assay 110
• 2) Determination of intercellular reactive oxygen species 112
• 3) DNA fragmentation patterns in CCRF S-180 II cells of oxidation 114
• 4) Protein analysis with western blot 116
• 5) Reverse transcription(RT)-PCR and Real time PCR 126
• 11. Effect of ellagic acid on the thickness of the epidermis 128
• 1) Effect of ellagic acid on the thickness of the epidermis 128
• 2) Histological observations 131
• 12. Result of stability test of liquid crystal 132
• 1) Microscopy analysis 132
• 2) X-rays diffraction (XRD) 139
• 3) Transmission electron microscope (TEM) 140
• 13. Result of stability test of cosmetics 142
• 1) Measurement of pH 142
• 2) Measurement of viscosity 144
• 3) Incubation (0, 25, 40 ℃) test 145
• 4) Cycle chamber and freeze thaw cycling test 146
• 5) Photo (Natural light) stability 148
• 14. Result of Safety test of cosmetics 149
• 1) Patch test of human skin 149
• Ⅳ. Discussion 150
• Ⅴ. References 151