Collagen 유래 펩타이드의 합성과 생체 효능비교
Preparation of collagen-derived peptides and efficacy of biomedical application
- 주제(키워드) Solid Phase Peptide Synthrsis , Collagen peptide , Peptide activity test
- 발행기관 아주대학교
- 지도교수 김문석
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위명 석사
- 학과 및 전공 산업대학원 화학생명공학과
- 실제URI http://www.dcollection.net/handler/ajou/000000017605
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
현재 펩타이드는 기능성 화장품 또는 의약품에 널리 사용되고 있다. 주 름개선, 피부노화방지에도 많이 적용되고 있으며, 또한 아토피와 여드름과 같은 치료의 목적으로도 많은 제품이 나오고 있다. 하지만 펩타이드는 일반적으로 사용되는 다른 화장품 원료에 비해 비싼 원료이다. 이로 인해 유효한 정도로 사용하기에는 비용부담이 높다. 펩타 이드의 가격은 아미노산의 수에 따라 또는 순도에 따라 가격이 다르게 책 정된다. 예전에는 높은 가격으로 사용이 제한되었으나, 현재는 국내에서 알려진 펩타이드 생산업체는 펩트론, 에이엔펩, 애니젠, 케어젠 등이 있다. 현재 펩타이드는 비교적 쉽게 접할 수 있으며 펩타이드를 활용한 여러 기능성 제품이 나오고 있다. 하지만 화장품이나 의약품에 펩타이드를 적용하기 위해서는 가격 경쟁력이 반드시 필요하다. 적은 비용으로 많은 펩타이드 를 생산할 수 있어야만 펩타이드 공급단가를 낮출 수 있다. 공급단가가 낮아지면 많은 소비자들이 낮은 가격으로 훌륭한 펩타이드 화장품, 의약 품을 접할 수 있을 것이다. 펩타이드 합성방법은 현재까지는 고체상법이 대표적이라 할 수 있다. 고 체상법에 사용되는 시약이나 용매는 수 없이 많다. 각 펩타이드별로 서로 다른 서열을 가지고 합성 된다. 펩타이드 특성은 서열에 따라 달라지며 사용되는 시약, 용매 또한 펩타이드의 품질이 달라질 수 있다. 따라서 본 연구는 Collagen 유래 펩타이드 중 효능이 우수한 한가지 펩 타이드를 선정하여 그 펩타이드에 대한 시약 및 용매의 변화를 통하여 고 순도 펩타이드를 합성하는 것에 목적이 있다. 현재 고체상법에 적용할 수 있는 여러 가지 알려진 Coupling Solvent (DMF, DMSO, DMA, THF, NMP) 및 Coupling Reagent (HOBt, HOOBt, HOAt, HBTu, HCTu, TBTu) 선정하여 Collagen 유래 펩타이드 의 최적화 합성방법을 연구하였으며, 실험결과 Coupling Solvent는 DMF/DMSO (7:3), Coupling Reagent는 HOBt/TBTu가 가장 고 순도의 펩타이드를 합성할 수 있었다. 합성된 펩타이드의 효능 비교로 Cell Activity (Keratinocyte, Fibroblast) 통해 관찰 하였으며, 고 순도로 합성 이 완료된 펩타이드를 통해 Cell Morphology와 Collagen Expression을 통 하여 Skin Cell 에서의 Anti-aging의 효과를 확인 하였다.
more초록/요약
Currently, Peptide is commonly used for functional cosmetics and medicine. It is also utilitized for anti-skin aging as well as anti wrinkle treatments. There are also many products for anit-ance and anti-atopy treatments. However, peptide is considered expensive compared to other cosmetics materials used in general. As a result, it costs too much to produce considering the level of effectiveness. Unlike the past when the usage of peptide was limited due to its high cost, there are a number of manufacturers who produce peptide such as peptron, anpep, anygen, caregen. Nowadays peptide is easily found and there is a wide selection of functional cosmetics products with peptide. Though, it is inevitable to have cost advantage to utilize peptide for cosmetics or medicines. In order to have the selling price of peptide lower, it should be produced at a lower cost. The lower the selling price of peptide is, the more consumers can afford to experience the effect of cosmetics and medicines with peptide. The most generally used peptide synthesis method would be SPPS. There are countless reagents or solvents used in SPPS. Each peptide is synthesized with different sequence. The characteristics of peptide could vary based on the sequence and the sequence also influences the quality of reagents, solvents as well as peptide. The purpose of this study is to select the most effective peptide among peptides derived from collagen and to synthesize high-purity peptide by coupling different solvents and reagents. We have researched to optimize synthesis method of collagen-derived peptide by coupling different solvents (DMF, DMSO, DMA, THF, NMP) and reagents (HOBt, HOOBt, HOAt, HBTu, HCTu, TBTu). As a result, we found out that the highest level of peptide purity was achieved under the combination of two solvents DMF/DMSO with the ratio of 7:3 and two reagents which are HOBt/TBTu. When peptide synthesized with high purity was injected into keratinocyte and fibroblast, cell activity was enhanced. Also anti-aging effect was confirmed by cell morphology and collagen expression.
more목차
논문 요약
Abstract
표 목차
그림 목차
Ⅰ. 서 론······················································································1
1. 펩타이드 ···········································································································1
2. 펩타이드 화장품 및 의약품 시장동향 ·······················································3
Ⅱ. 실험재료 및 방법 ·····························································································7
1. 펩타이드 합성 ·································································································7
2. 펩타이드 분석 ·································································································8
2.1 HPLC Analysis ··························································································8
2.2 LC/Mass Analysis ····················································································9
3. 펩타이드 Activity Test ···············································································9
3.1 Fibroblast를 이용한 Cell Activity Test ··············································9
3.2 Keratinocyte를 이용한 Cell Activity Test ·······································10
3.3 Cell Morphology Test ············································································10
3.4 Collagen Expression Test ·····································································10
III. 결과·····················································································13
1. 펩타이드 Screening ···························································13
1.1 Collagen 유래 펩타이드 선정 ·······························································13
1.1.1 HPLC 분석결과···················································································14
1.1.2 Cell Activity 결과 ··············································································15
2. 펩타이드 합성법 변경에 의한 Purity 및 Activity 결과 ·····················17
2.1 합성 Solvent 변화에 따른 결과 ························································· 17
2.1.1 Purity의 변화···················································································· 18
2.1.2 Cell Activity의 변화 ······································································ 21
2.2 합성 Reagent 변화에 따른 결과 ·························································· 24
2.2.1 Purity의 변화 ······················································································ 25
2.2.2 Cell Activity의 변화········································································ 27
3. 선정된 펩타이드의 항노화 및 항산화 기능 검증 ··································· 29
3.1 Cell Proliferation Test ············································································· 29
3.2 Cell Morphology Test ·············································································· 29
3.3 Collagen Expression 확인 ······································································· 29
Ⅳ. 결론 ·····················································································30
1. 선정 펩타이드의 Optimzation 된 합성조건 ··········································· 30
2. 선정 펩타이드의 Anti-aging으로의 Effect ··········································· 30
3. 선정 펩타이드의 적용 ················································································· 31
참고 문헌······················································································32
