Studies on Multi-Functional Cosmetics Activities of Ganoderma lucidum Fermentation Broths
- 주제(키워드) 영지 균사체 발효물 , 화장품 , 항노화 , 미백
- 발행기관 아주대학교
- 지도교수 민철기
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 박사
- 학과 및 전공 일반대학원 응용생명공학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030919
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
This study was designed to find and examine any cosmetic activity of the Ganoderma. lucidum (G. lucidum） fermentation broths obtained by submerged fermentation method with respect to G. lucidum strain screening, the optimization of fruiting body fermentation process, the identification of essential metabolites, the measurement of anti-oxidation activity and whitening activity, and their cosmetic efficacy evaluation on the human skin. Compared to the extract from its fruiting body and other parts of G. lucidum, its fruiting body fermentation broths have some advantages in short production cycle, in process control, and in low-cost. As new cosmetic raw materials replacing the traditional method utilizing, for example, the G. lucidum fruiting body, the G. lucidum fermentation broths reduce production costs but improve the quality of products, hence is of great significance in the field of cosmetics. The contents of total saccharides, triterpenes, and total phenols, and antioxidant and tyrosinase inhibition activities of the G. lucidum fermentation broths were higher than those of either ethanol or hot water extracts from the fruiting body. The antioxidant and tyrosinase inhibition activities were strongly affected by the cultivation conditions. The best fermentation medium was malt medium, and the fermentation period was 5–6 days. Under such conditions, the rates of DPPH radical scavenging, superoxide anion scavenging, and tyrosinase inhibition can be maintained at 94.05%, 30.54%, and 93.08%, respectively, compared to the control, indicating that the G. lucidum fermentation broth is an effective source of naturally active ingredients, thus has a good health care application prospect. With the G. lucidum fermentation broth, the mycelium, the fruiting body and the spores, essential metabolites were measured. A total of 48, 25, 364 and 28 metabolites were detected in each fermentation broth, respectively. 15 and 9 polysaccharides were up-regulated in the fermentation broth and the mycelium extracts, respectively. In addition, triterpenoids were detected predominantly in the fruiting body and betaine mainly were detected in the spore. Besides, this study provided a comprehensive description of overall metabolic landscape of fermentation broth, the mycelium, the fruiting body and the spores, especially the fermentation broth, which would facilitate the studies on the function and pharmacodynamics of different parts of G. lucidum at the molecular level. The G. lucidum fruit extracts and the G. lucidum fermentation broths also manifested a significant cytotoxic activity against malignant skin cells but a low cytotoxic activity against normal skin cells in vitro. A significant difference in its cytotoxic activity was observed between the tested extracts and the G. lucidum fermentation broths. The above results may indicate that the G. lucidum fermentation broths provide effective and safe sources for cosmetic ingredients and thus have good application prospects in the R&D of cosmetics. To further verify the cosmetic advantage of the G. lucidum fermentation broth, a comparison of cosmetic function between the fruiting body ethanol extract and its fermentation broths were made by in vitro experiment. Skin anti-aging effects of the fruit ethanol extracts and the fermentation broths were evaluated in relation to normal human dermal fibroblasts with respect to the expression of MMPs and TIMP-1 after cultured in the presence of the fruit ethanol extracts or the fermentation broths at 100 ppm. The G. lucidum fermentation broths down-regulated the expression of MMP-1, MMP-3, MMP-9 by 61.59%, 39.21%, 49.49% of control, respectively while they up-regulated the expression of TIMP-1 by 151.95% of control. In addition, the fermentation broths up-regulated the expression of procollagen Ⅰ secretion in UVB-irradiated (40 mJ/cm2) NF cells by 139.92% over control, indicating that the G. lucidum fermentation broths are good sources for skin anti-aging cosmetics. The G. lucidum fermentation broths down-regulated the activity of cellular tyrosinase at 42.76% of control, exhibiting cellular tyrosinase inhibition of 57.24%. Additionally, the G. lucidum fermentation broths also inhibited the melanin production in B16F10 cells. The inhibitory efficacy of total melanin production in B16F10 cells treated with the G. lucidum fermentation broths at 100 ppm were at the value of 30.60% of sham non-treated cells. For further cosmetic evaluation of the G. lucidum fermentation, we manufactured a skin care cream containing the G. lucidum fermentation broth and measured its cosmetic efficacies in various categories. The skin cream increased skin hydration, decreased skin trans-epidermal water loss (TEWL), reduced (small/medium) fine lines, (small/medium) wrinkles, melanin index, melanin concentration, skin pigmentation area significantly after 4 weeks of topical use. It also significantly increased the skin texture roughness and decreased the melanin production. In summary, the G. lucidum fermentation broths show stronger anti-aging and whitening effects than the G. lucidum fruiting body ethanol extracts. It is suggested that the G. lucidum fermentation broths are safe and efficient in many skin care functions, implying that these unique metabolites are promising in new skin care cosmetics development.
more목차
CHAPTER 1. General introduction 20
1.1 Ganodoma lucidum and its nutrients 20
1.2 Chemical constituents of Ganoderma lucidum 21
1.2.1 Polysaccharides 21
1.2.2 Triterpenes 22
1.3 Biological activities 24
1.3.1 Antioxidant activities 25
1.3.2 Whitening efficacy activity of G. lucidum 25
1.4 G. lucidum fermentation broths and their bioactivities 26
1.5 Research objects 26
Chapter 2. Comparative study on bioactivities from G. lucidum body extraction and fermentation broths 28
2.1 Introduction 28
2.2 Materials and mathods 31
2.2.1 Microorganisms 31
2.2.2 Optimization of extraction conditions to obtain active components from G. lucidum fruiting body 31
2.2.3 Optimization of G. lucidum submerged fermentation condition 32
2.2.4 Total saccharide and triterpene content detection 32
2.2.5 Determination of total phenol content 33
2.2.6 Detection of Antioxidatioan Activity of Fermentation Broth and Extract 33
2.2.7 Detection of tyrosinase activity inhibition 36
2.3 Statistical analysis 36
2.4 Results 36
2.4.1 Extraction of active components from G. lucidum fruiting body 36
2.4.2 Optimization of G. lucidum submerged fermentation conditions 41
2.4.3 Bioactivity comparison between ethanol extract and fermentation broth 44
2.5 Discussion and Conclusion 47
Chapter 3. Untargeted metabolomics of G. lucidum body extracts and fermentation broths 51
3.1 Introduction 51
3.2 Materials and methods 52
3.2.1 G. lucidum information 52
3.2.2 Sample preparation 53
3.2.3 LC-MS analysis 53
3.2.4 LC-MS/MS data processing and multivariate analysis 54
3.2.5 Metabolite correlation network construction 54
3.3 Results 55
3.3.1 Metabolic profile of the fermentation broth, the mycelium, the fruiting body and the spore powder 55
3.3.2 Principal component analysis (PCA) and supervised OPLS-DA reveals differential metabolites in the fermentation broth, the mycelium, the fruiting body and the spores 58
3.3.3 Characteristic features of G. lucidum triterpenoids in the fermentation broth, the mycelium, the fruiting body and the spore 63
3.3.4 Characteristic features of G. lucidum polysaccharide and nucleosides in fermentation broth, mycelium, fruiting body and spores powder 65
3.3.5 Characteristic features of G. lucidum alkaloids in fermentation broth, mycelium, fruiting body and spores powder 68
3.3.6 Major differential metabolic pathways among fermentation broth, mycelium, fruiting body and spores 71
3.3.7 Metabolites correlation analysis among fermentation broth, mycelium, fruiting body and spore 74
3.4 Conclusion 75
Chapter 4. Cytotoxic activity of the G. lucidum body extracts and the fermentation broths 76
4.1 Introduction 76
4.2 Materials and mathods 77
4.2.1 G. lucidum information 77
4.2.2 Sample preparation 77
4.2.3 cytotoxicity activity 77
4.3 Results 78
4.4 Conclusion 80
Chapter 5. Skin anti-aging effects of the G. lucidum fruiting body extracts and the fermentation broths 81
5.1 Introduction 81
5.1.1 Skin aging mechanisms 81
5.1.2 Mechanism related to collagen biosynthesis 84
5.1.3 Mechanism related to MMPs and TIMPs 85
5.1.4 ECM- cell interaction 87
5.1.5 Cytokines in skin aging 88
5.1.6. Antioxidant enzymes 88
5.2 Materials and methods 90
5.2.1 G. lucidum information 90
5.2.2 Sample preperation 90
5.2.3 Cell culture 91
5.2.4 MTT assay on normal human dermal fibroblasts 91
5.2.5 Skin anti-aging of G. lucidum extracts and the fermentation broths 92
5.2.5.1 UVB irradiation and the body extract and the fermentation broth treatment 92
5.2.5.2 Inhibitory effects of the G. lucidum extract and the fermentation broths on the expression and the activity of MMPs and TIMPs 92
5.2.6 Statistical analysis 93
5.3 Results 93
5.3.1 MTT assay results on normal human dermal fibroblasts 93
5.3.2 UVB irradiation effects of the G. lucidum extracts and the fermentation broths on NF cells viability 94
5.3.3 Inhibitory effects of the G. lucidum extract and the fermentation broths on the expression and the activity of MMPs and TIMPs 96
5.4 Conclusion 100
Chapter 6. Skin whitening effects of the G. lucidum fruiting body extracts and the fermentation broths 102
6.1 Introduction 102
6.1.1 Skin whitening mechanisms 102
6.1.2 Down regulation of proteins related with transcriptional regulation of melanogenic enzymes 104
6.1.3 Inhibition of post-translational modification of melanogenic enzymes (melanosome biogenesis) 104
6.1.4 Sorting of melanosomes in melanocytes 105
6.1.5 Transfer of melanosomes to the tip of melanocyte dendrites and finally into keratinocytes 106
6.2 Materials and mathods 106
6.2.1 G. lucidum information 106
6.2.2 Sample preperation 107
6.2.3 Skin whitening effects of fruiting body extracts and fermentation broths 107
6.2.3.1 Cell Culture 107
6.2.3.2 MTT assay on mouse melanoma B16F10 cells 108
6.2.3.3 Measurement of cellular tyrosinase activity in B16F10 cells 108
6.2.3.4 Measurement of melanin contents of B16F10 cells 109
6.2.5 Statistical analysis 109
6.3 Results 110
6.3.1 MTT assay on mouse melanoma B16F10 cells 110
6.3.2 Effects of the G. lucidum extracts and the fermentation broths on tyrosinase activity of B16F10 cells 110
6.3.3 Effects of the G. lucidum extracts and the fermentation broths on melanin contents of B16F10 cells. 112
6.4 Conclusion 113
Chapter 7. Cosmetic efficacy evaluation of the G. lucidum fermentation broths 114
7.1 Introduction 114
7.2 Materials and mathods 115
7.2.1 Instruments and evalution methods 115
7.2.2 Preparation of the G. lucidum fermentation broths ingradients 116
7.2.3 Cosmetic formulation and manufacturing 116
7.2.4 Requirements for measurement conditions and volunteers 118
7.2.5 Topical application and evaluation 119
7.2.6 Statistical analysis 120
7.3 Results 121
7.3.1 Volunteer Statistics 121
7.3.2 Skin anti-aging effects of the G. lucidum fermentation broth-containing cream 121
7.3.2.1 Measurement of TEWL 121
7.3.2.2 Skin cuticle hydration test 122
7.3.2.3 Measurement of the skin elasticity 122
7.3.2.4 Measurement of wrinkles 125
7.3.3 Skin whitening effects of G. lucidum fermentation broths 132
7.3.3.1 Measurement of skin color 132
7.3.3.2 Measurement of pigmentation 133
7.4 Conclusion 136
Chapter 8. General discussion and conclusion 137
8.1 Bioactivity components in the G. lucidum body extracts and the fermatation broths 137
8.2 Bioactivity of the G. lucidum body extraction and the fermatation broths 138
8.3 Cytotoxic activity of the G. lucidum body extract and the fermentation broths 139
8.4 Multi-functional cosmetic activities of the G. lucidum body extracts and the fermentation broths 139
Referance 141
