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밀순 효소처리추출물이 미백 및 주름 개선에 미치는 영향

Effects of enzyme-treated Triticum aestivum sprout extracts on the activity of whitening and anti-wrinkle

초록/요약

현대사회는 환경오염으로 인해 자외선에 의한 피부 손상도가 증가하고 있으며 피부노화, 피부색의 변화와 색소 침착을 가중시키고 있다. 뿐만 아니라 미용적인 이유에서 피부 색소침착에 대한 관심은 증가하고 있으며, 이러한 요구에 발맞춰 미백 소재 개발 연구가 활발히 진행되고 있다. 본 연구에서는 밀순 추출물(TA)과 효소처리추출물(ETA)을 이용하여 화장품 소재로서의 가능성을 확인하기 위해 항산화 활성, 미백 및 주름 개선 효과를 관찰하고 그 작용기전을 검증하였으며, 발효추출물의 활성 성분을 분석하였다. 그 결과 α-MSH로 자극한 그룹에 비해 ETA 추출물을 처리한 그룹에서 tyrosinase 활성이 감소한 것을 확인하였고, 멜라닌 함량 또한 농도 의존적으로 ETA 추출물에서 감소한 것을 확인하였다. 미백관련 인자들을 realtime-PCR과 western bolt으로 분석한 결과 tyrosinase, TRP-1, TRP-2 및 MITF의 발현을 모두 유의적으로 억제하는 것을 확인하였다. 주름개선 효과를 확인하기 위해 elastase, collagenase, MMP-1 및 procollagen 활성 분석을 실시한 결과 UVB를 조사한 그룹에 비해서 ETA 추출물이 유의적인 효과를 나타내었다. 본 실험을 통하여 ETA 추출물이 tyrosinase, melanin 활성을 저해시켰으며, 이를 통해 tyrosianse, TRP-1, TPR-2 그리고 MITF 발현을 효과적으로 저해한다는 사실을 확인하였고, elastase, collagenase 및 MMP-1, procollagen의 활성 또한 유의적으로 억제시키는 것을 확인하여 기능성 미백 및 주름개선 화장품 소재로서의 활용 가능성을 제시하였다.

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초록/요약

Melanogenesis is the bioprocess of melanin production by melanocytes in the skin and hair follicles and is mediated by several enzymes, such as tyrosinase, tyrosinase related protein (TRP)-1, TRP-2, and MITF. In this study, it was investigated the effect of Triticum aestivum sprout (TA) and enzyme-treated extracts (ETA) on tyrosinase activity, melanin production and collagenase activity et al. as natural products of whitening and anti-wrinkle functional cosmetics. The inhibition of tyrosinase activity of ETA significantly increased of tyrosinase activity compared to the α-Melanocyte stimulating hormone (α-MSH) treated group. In addition, Melanin content in murine B16F10 melanoma cells were decreased by ETA extracts in the dose-dependent manner. Among the enzyme-treated extracts, ETA 50% EtOH was especially effective. Furthermore, western blot analysis revealed that ETA extracts significantly downregulated the expression of tyrosinase, TRP-1, TRP-2 and MITF which treat of α-MSH–induced melanogenesis in murine B16F10 melanoma cells. Furthermore, elastase activity and collagenase activity inhibition were decreased by ETA extracts in a dose-dependent manner. And, the amount of type Ⅰ procollagen, which is a precursor of collagen, was also increased as compared with the UVB-treated group. In conclusion, these results showed that ETA can inhibit melanin synthesis through inhibitory action on tyrosinase activity and inhibition elastase activity, suggesting that these results can be used for the study on maintaining skin elasticity and whitening.

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목차

Ⅰ. INTRODUCTION 1

Ⅱ. MATERIALS AND METHODS 5
2.1. MATERIALS 5
2.2. Preparation of Triticum aestivum extracts 6
2.3. Preparation of enzyme-treated Triticum aestivum sprout extracts 7
2.4.1 DPPH radical scavenging activity 9
2.4.2 ABTS+ radical scavenging activity 9
2.5.1 Mushroom tyrosinase activity assay 11
2.5.2 Cell culture 11
2.5.3 Cell viability 11
2.5.4 Melanin content assay 12
2.5.5 Intracellular tyrosinase activity assay 13
2.5.6 Realtime-polymerase chain reaction (Realtime-PCR) 14
2.5.7 Western blot Analysis 15
2.6.1 Elastase activity assay 17
2.6.2 Collagenase activity assay 17
2.6.3 Cell culture 18
2.6.4 UVB irradiation 18
2.6.5 Cell viability 19
2.6.6 MMP-1 activity assay 19
2.6.7 Type Ⅰ procollagen assay 20
2.7. High Performance Liquid Chromatography (HPLC) analysis 22
2.8. Statistical analysis 23

Ⅲ. RESULTS 24
3.1. Anti-oxidant effect
3.1.1 Effects of ETA on DPPH radical scavenging activity 24
3.1.2 Effects of ETA on ABTS+ radical scavenging activity 26
3.2. Whitening effect
3.2.1 Effects of ETA on mushroom tyrosinase activity 28
3.2.2 Effects of TA and ETA on cell viability in B16F10 mouse melanoma cells 30
3.2.3 Effects of ETA on melanin content in B16F10 cells 32
3.2.4 Effects of ETA on intracellular tyrosinase activity in B16F10 cells 34
3.2.5 Effects of ETA on mRNA expression rate in B16F10 cells 36
3.2.6 Effect of ETA extract on melanogenesis-related proteins expression 41
3.3. Anti-wrinkle effect
3.3.1 Effects of TA and ETA on elastase inhibition activity 46
3.3.2 Effects of TA and ETA on collagenase inhibition activity 48
3.3.3 Effects of TA and ETA on cell viability in HS68 cells 50
3.3.4 Effects of ETA on UVB-induced secretion of MMP-1 in HS68 cells 52
3.3.5 Effects of ETA on UVB-induced secretion of type Ⅰ procollagen in HS68 cells 54
3.4. Contents analysis
3.4.1 α-linolenic acid content variation of TA and ETA by HPLC analysis 56
3.4.2 Isoorientin and isoscoparin content variation of TA and ETA by HPLC analysis 59

Ⅳ. DISCUSSION 62

Ⅴ. REFERENCE 68

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