Studies on multi-functional cosmetics activities of peptide hydrolysates from cultured plant cells of Vigna radiata L.
Studies on multi-functional cosmetics activities of peptide hydrolysates from cultured plant cells of Vigna radiata L.
- 주제(키워드) peptide hydrolysate , Mungbean , two dimensional electrophoresis , skin anti-aging , skin-whitening
- 발행기관 아주대학교
- 지도교수 변상요
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위명 석사
- 학과 및 전공 일반대학원 응용생명공학과정
- 실제URI http://www.dcollection.net/handler/ajou/000000020481
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
This study was subjected on Mungbean (Vigna radiata L.) peptide hydrolysates that derived from the suspension culture cell. Extraction of proteins from Mungbean powder had been evaluated and gotten the optimal condition consist of using Lysis buffer contained 62.5mM Tris, 10% glycerol and 0.5% SDS and adjust pH to 8. Peptide hydrolysates were prepared via though enzymatic method of combination trypsin and chymotrypsin at ratio of substrate : enzymes as 25:1:1 (w/w/w). Crude peptide hydrolysates suspension was isolated on 40% polyacrylamide gel electrophoresis into the fractions that owned peptides in difference from MW range. The bigger peptides focused on the top of gel and smaller peptides presented on the bottom of gels. Loading peptide standards together with Mungbean peptide hydrolysates and separated on electrophoretic gel, the results found that peptides having MW of around 1000, 500-600 and 200-300 Da focused on fraction 5, fraction 8 and fraction 10, respectively. These results suggested that fraction 5 to 10 focused peptide hydrolysates possessing MW around 1000 Da and less than. Skin anti-aging efficacy of Mungbean peptide hydrolysates was evaluated via though proteomics analysis. Proteins of Normal Human Dermal Fibroblasts treated and non-treated with peptide hydrolysates fractions were analyzed according to 5 mechanisms of skin anti-aging. Results found that Mungbean peptide hydrolysates are the good sources of skin anti-aging. The best efficacy belong on fraction 6 that up-regulated the expression of enzymes of collagen synthesis as P4H1 (111.4%), P4H2 (295.6%), P3H1 (111.7%), P3H2 (106.5%), LH3 (534.9%), PDI (107.5%) and PDIA2 (263.0%); up-regulated the expression of TIMP-1 (859.2%); up-regulated the expression of receptor in ECM-cell linkage as integrin β3 (228.8%); up-regulated the expression of endogenous antioxidant enzymes including GSTA1 (271.5%), GPX2 (178.8%), Catalase (109.1%), GSH (125.7%) and GSTM3 (265.1%). Moreover, Mungbean peptide hydrolysates of fraction 6 down-regulated the expression of MMPs including MMP-1 (33.3%), MMP-2 (61.9%), MMP-3 (49.1%), MMP-8 (32.8%) and MMP-13 (47.6%); down-regulated the expression of cytokines as IL-1β (90.6%), IL-6 (66.5%), NF-κβ (73.2%) and TNF-α (44.4%). Additional, proteomics analysis on proteins of Mouse melanoma B16F10 treated and non-treated with Mungbean peptide hydrolysates fractions observed that Mungbean peptide hydrolysates down-regulated the expression of signal, transfer and enzyme proteins in 4 mechanism of skin whitening. Therefore, these peptides are the good sources for skin whitening. The best efficacy was showed on peptides of fraction 7. Detail, these peptides down-regulated the expression of proteins in transcription of melanogenesis as ACTH-R (54.8%), CREB-1 (19.5%), MITF (35.9%), SCF (15.7%); down-regulated the expression of proteins in post-transcriptional modifications including PKC-β (51.2%), TYRP-2 (9.5%), TYR (90.6%); down-regulated the expression of proteins in sorting of melanosomes and in transfer of melanosomes to melanocyte dendrite tips and to keratinocytes as PMEL (25.6%) and (FOXN1 (43.9%), respectively. Otherwise, the inhibition of melanin production was demonstrated on B16F10 treated with peptide hydrolysates from Mungbean. All of Mungbean peptide fractions inhibited melanin production. The best efficacy was observed on cell treated with peptides of fraction 7 (81.4% of control). These results suggested that Mungbean peptide hydrolysates are the good components for skin whitening.
more목차
I. INTRODUCTION 1
A. Mungbean and its nutrients 1
B. Chemical constituents of Mungbean 2
C. Biological activities 3
1. Antioxidant activities 3
2. Antimicrobial activity 4
3. Anti-inflammatory activity 4
D. Peptide hydrolysates and their bioactivities 5
E. Peptide hydrolysates from mungbean 7
F. Skin aging mechanisms 9
1. Mechanism related with collagen biosynthesis 10
2. Matrix Mellanoproteinases (MMPs) and Tissue inhibitor of Matrix mellanoproteinases (TIMPs) 11
3. ECM- cell interaction 12
4. Cytokines in skin aging 13
5. Antioxidant enzymes 13
G. Skin whitening mechanisms 14
1. Down regulation of Proteins related with Transcriptional Regulation of Melanogenic Enzymes 17
2. Inhibition Post-Translational Modification of Melanogenic Enzymes (melanosome biogenesis) 17
3. Sorting of melanosomes in melanocytes 18
4. Transfer melanosomes to the tip of melanocyte dendrites and finally transfer into keratinocytes 19
II. MATERIALS & METHODS 20
A. Materials & intruments 20
B. Methods 21
1. Optimization of extract method 21
2. Preparation, fraction and purification of Mungbean peptide hydrolysates 21
3. Skin anti-aging investigation of Mungbean peptide hydrolysates on Normal Human Dermal Fibroblasts 22
4. Skin whitening investigation of Mungbean peptide hydrolysates on B16F10 24
III. RESULTS & DISCUSSIONS 26
A. Optimization of protein extraction from Mungbean powder 26
1. Results on pH optimization of protein extraction 26
2. Result on choose the method for cell wall disruption 27
B. MS analysis 28
C. Result on skin anti-aging of Mungbean peptide hydrolysates 36
1. MTT test results on Normal Human Dermal Fibrolasts 36
2. Skin anti-aging of Mungbean peptide hydrolysates via proteomics analysis 38
2.1. Effects of Mungbean peptide hydrolysates to expression of enzymes in collagen synthesis 40
2.2. Effects of Mungbean peptide hydrolysates to MMPs expression 43
2.3. Effect of Mungbean peptide hydrolysates to expression of proteins related ECM-cell interaction 45
2.4. Effect of Mungbean peptide hydrolysates to the expression of cytokines. 47
2.5. Effect of Mungbean peptide hydrolysates to expression of antioxidant enzymes 49
D. Results on skin whitening effects of Mungbean peptide hydrolysates 53
1. MTT test on mouse melanoma B16F10 53
2. Skin whitening of Mungbean peptide hydrolysates via though proteomic test 55
2.1. Transcription of melanogenetic proteins 57
2.2. Post-transcriptional modification 59
2.3. Sorting of melanogenetic proteins in melanosomes 61
2.4. Transfer melanosomes to tip of melanocytes dendrites and into keratinocytes 62
3. Results on melanin producing inhibition of Mungbean peptide hydrolysates 64
CONCLUSIONS 65
REFERENTS 67
