Title Page
Contents
Abstract 13
CHAPTER I. Introduction 17
1. Background 18
2. Objectives 19
3. Scope and Limitations 20
CHAPTER II. Review of Related Literature 21
1. Nitric oxide 22
1.1. Nitrogen cycle 22
1.2. Physicochemical properties and biological activity of NO 22
1.3. Duality character of NO 23
1.4. Biosynthesis of NO 24
1.5. NO donor drugs 25
2. Oxidative stress 26
2.1. Free radical 26
2.2. Ultraviolet radiation and oxidative stress 27
3. Metabonomics 28
3.1. Application of metabolomics in medicine 28
3.2. Natural plant metabonomics 29
4. Reference 31
CHAPTER III. Optimization of the extraction method of adherent cell metabolites and application on UVB-irradiated HaCaT and B16 cell line photoaging 43
1. Introduction 44
2. Material and Method 45
2.1. Reagent 45
2.2. Cell culture 45
2.3. Design of experiments 46
2.4. Application of metabolite extraction protocol in photoaged HaCaT and B16 cells 49
2.5. Metabolites analysis 50
2.6. Statistical analyses 50
3. Results and discussion 51
3.1. Optimization of extraction scheme 51
3.2. Effects of UVB irradiation on theactivity of HaCaT and B16 cells 52
3.3. Precision analysis 52
3.4. Significant difference metabolite analysis 53
3.5. PCA and Heatmap 54
4. Conclusion 56
5. Reference 57
CHAPTER IV. Exogenous nitric oxide promotes wound healing in in vitro models of HaCaT 74
1. Introduction 75
2. Materials and methods 77
2.1. Cell culture and grouping 77
2.2. MTT cell viability assay 77
2.3. Flow cytometry 78
2.4. Detection of oxidative stress level 78
2.5. Determination of MMP-2 and MMP-9 in the supernatant of HaCaT cells using ELISA 79
2.6. in vitro Wound healing assay 79
2.7. Metabolomics analysis 79
2.8. Statistical analysis and Metabolic network analysis 80
3. Results and analysis 80
3.1. MTT Assay 80
3.2. NO and MDA production 81
3.3. MMPs-2,9 assay 81
3.4. NO induce cell proliferation 82
3.5. in vitro cell scratch healing assay 83
3.6. Metabolite identification and analysis 83
4. Discussion 84
4.1. Cell activity and oxidative stress 84
4.2. Cell cycle 84
4.3. MMPs 85
4.4. Biochemical interpretation 86
5. Conclusion 88
6. Reference 89
CHAPTER V. Comparison of antioxidant properties and metabolite profiling of Acer pseudoplatanus leaves of different colors 104
1. Introduction 105
2. Materials and Methods 106
2.1. Maple leaves 106
2.2. Determination of polyphenol content 107
2.3. In vitro Antioxidant test 109
2.4. Extraction and analysis of metabolites 112
2.5. Statistical analysis and Metabolic network analysis 113
3. Results 113
3.1. Polyphenol content 113
3.2. Antioxidant activity 114
3.3. Correlation coefficients of antioxidant activities and polyphenols content 116
3.4. Metabolite profiling 117
4. Discussion 118
5. Conclusion 120
6. References 122
CHAPTER VI. Anti-UVB radical effects of Acer pseudoplatanus leaves extract on HaCaT cells 142
1. Introduction 143
2. Materials and Methods 144
2.1. Maple leaves 144
2.2. Determination of polyphenolcontent and GC/MS base metabolites analysis 144
2.3. DPPH free radical scavenging effect 145
2.4. Evaluation of maple leaves antioxidant capacity at cell level 145
2.5. NO generation and lipid peroxidation (MDA) 146
2.6. Matrix metalloproteinases assay 147
2.7. Flow cytometry analysis 147
2.8. Extraction and analysis of metabolites 148
2.9. Statistical analysis and Metabolic network analysis 149
3. Results and Discussion 149
3.1. Determination of the polyphenol content and DPPH free radical scavenging effect 149
3.2. Cell viability 150
3.3. Oxidative stress 150
3.4. MMP-2,9 152
3.5. Metabolite profiling 152
4. Discussion 153
4.1. Cell activity and oxidative stress 153
4.2. Biochemical interpretation 155
5. Conclusion 156
6. Reference 157
CHAPTER VII. Conclusions 173
1. Result 174
2. Outlook and Limitation 174
CHAPTER III 10
Table 3-1. Selection of extractant. 63
Table 3-2. Selection of quenching and harvesting methods. 64
Table 3-3. Effects of different irradiation doses on the activity of HaCaT and B16 cells. 65
Table 3-4. RSD of differential metabolites contained in both Hacat and B16 cells. 66
Table 3-5. Variation trends of differential metabolites contained in both Hacat and B16 cells. 67
CHAPTER IV 10
Table 4-1. Differential metabolite List. 96
CHAPTER V 10
Table 5-1. Total content of different polyphenols. 138
Table 5-2. List of identified metabolites. 139
Table 5-3. List of twenty metabolites with high absolute loading values on PC1 and PC2. 141
CHAPTER VI 10
Table 6-1. Yield, Polyphenol content, and In vitro Antioxidant capacity. 170
Table 6-2. Differential metabolite List. 171
CHAPTER III 11
Figure 3-1. Extration scheme optimization flow chart. The metabolite extraction stage is divided into three parts in total, which are used to optimize the extractant, cell harvesting and quenching methods, and the effect of additional cell lysis on metabolite extraction. Images obtained... 68
Figure 3-2. Radar plot of metabolite extraction protocol optimization. (a) Screening of extractants, a total of 11 kinds of extractants were screened. (b) Screening of harvesting and quenching modes, a total of 12different combinations were evaluated. (c) Evaluation of additional cell... 69
Figure 3-3. VIP scores. PLS-DA analysis ofphotoaging metabolitesin two cell lines, obtained from Metaboanalyst. 70
Figure 3-4. Box chart of significant difference metabolites. Boxcharts of metabolite for the same trends in HaCaT and B16. Results were obtained by One-way ANOVA and fisher's LCD at Metaboanalyst. 71
Figure 3-5. PCA Scores Plot. The scores plot of the two cell lines was obtained by PCA at Metaboanalyst. 72
Figure 3-6. Heatmap. The heatmap of the two cell lines was obtained by PCA at Metaboanalyst. 73
CHAPTER IV 11
Figure 4-1. Cell viability with difference concentration of NO. Data represent the means ± SD percentage migration distance of sixes replicates (p〈0.05). 98
Figure 4-2. NO, MDA, MMP-2 and 9 content with difference concentration of NO. Data represent the means ± SD percentage migration distance of at least three replicates (p〈0.05). 99
Figure 4-3. Effect of NO on cell cycle of HaCaT cells 100
Figure 4-4. Effect of NO on cell migration. (a) Wound area changes after treatment with different NO doses. Data represent the means ± SD percentage migration distance of at least three replicates (p〈... 101
Figure 4-5. Principal component analysis (PCA) Plot of metabolites. PCA Plot obtained by metaboanalyst 5.0. 102
Figure 4-6. Plot of variable importance in projection (VIP) of metabolite. VIP Plot was obtained by metaboanalyst 5.0. 103
CHAPTER V 11
Figure 5-1. Maple leaves of different colors in Jeonju University. 133
Figure 5-2. In vitro antioxidant properties of maple leaves of different colors. (a) Dose- dependent TBARS absorbance values of maple leaves of different colors; (b) Dose-... 134
Figure 5-3. Correlation coefficients of antioxidant activities and polyphenol content. Correlation Plot was obtained by OriginPro 2021 (OriginLab Corporation, Northampton,... 135
Figure 5-4. PCA score plots. The PCA score plots were obtained by SIMCA (version 14.1, Umetrics, Umea, Sweden). Each point on the PCA score scatter plot represents a sample, and... 136
Figure 5-5. Metabolite heatmap with a dendrogram. 137
CHAPTER VI 11
Figure 6-1. Maple leaves of different colors. 163
Figure 6-2. Relative cell activity.(a) Cytotoxicity test of different doses of MLE. (b) Effect of MLE and UVB combined treatment on cell activity. 164
Figure 6-3. Effects of combined UVB and MLE treatment on NO, MDA, MMP-2, and MMP-9. 165
Figure 6-4. Effects of combined UVB and MLE treatment on ROS. 166
Figure 6-5. The effect of MLE in the process of programmed death in HaCaT cells after UVB treatment. The early apoptotic events (Annexin+/PI-) are shown in lower right quadrant... 167
Figure 6-6. PCA Score Plot. Red, containing 30 μg/mL RMLE in the medium, so as Yellow and Green. RUVB, after pretreatment of red maple leaves for 24 hours, the cells were... 168
Figure 6-7. Principal component analysis (PCA) Plot of metabolites. 169