Potential Effect of Multifunctional Transfersome Gel with Rhus chinensis Mill. Fruit and Bark Extract on Photo-aging Mechanisms

다국어 초록 (Multilingual Abstract)

This study investigates the anti-aging properties of Rhus chinensis Mill. fruit (RCF) and bark (RCB) extracts and their mixture (RCFB), focusing on their anti-wrinkle, antioxidant, anti-melanogenic, and anti-inflammatory activities. Anti-wrinkle efficacy was evaluated through elastase inhibition, elastase activity in UVB-induced human dermal fibroblast (HDF) cells, and mRNA expression of MMP-1, MMP-3, MMP-9, and COL-1. RCFB significantly reduced elastase activity, MMP expression, and increased COL-1 expression than the individual extracts. For anti- melanogenic activity, we assessed mushroom tyrosinase inhibition, intracellular tyrosinase activity, and melanin content in IBMX-stimulated B16F10 cells, alongside the mRNA expression of melanogenesis-related genes (MC1R, MITF, tyrosinase, TYRP-1, and TYRP-2). RCFB exhibited strong tyrosinase inhibition, decreased melanin production, and downregulated the expression of melanogenesis-related genes. Anti-inflammatory effects were determined by measuring chemokine content (TARC, MDC, IL-6, and TNF-α) using ELISA and the mRNA expression of the NF-κB pathway. RCFB effectively reduced chemokine levels and the expression of NF-κB, TARC, MDC, IL-6, TNF-α, and IL-8. Antioxidant activity was evaluated through total phenolic content (TPC), total flavonoid content (TFC), DPPH, and reducing power assays, along with reactive oxygen species (ROS) generation, CAT, SOD, GPx, and GST activities in H₂O₂-induced HaCaT cells, and Nrf2/Keap1 pathway expression. RCFB demonstrated high TFC, strong antioxidant activities, reduced ROS generation, increased antioxidant enzyme activities, decreased Keap1 expression, and upregulated SOD, CAT, GPx, and GST expression. To enhance phytochemical absorption and bioavailability, transfersomes (using soy phosphatidylcholine and Tween 80) loaded with RCFB (TRC) were synthesized. The optimized formulation (F4) exhibited 71.04 ± 0.45% entrapment efficiency, 246.75 ± 26.51 nm size, 0.21 ± 0.04 polydispersity index, and -28.5 ± 3.21 zeta potential. F4 was incorporated into a xanthan gum gel (XTRC), displaying higher spreading property, pH, stability, lower drug leakage, gradual drug release, and superior sun protection factor compared to TRC alone. TRC and XTRC showed enhanced anti-melanogenic activity by inhibiting tyrosinase, reducing melanin production, and downregulating the expression of tyrosinase, TYRP-1, TYRP- 2, MITF, and MC1R. It exhibited significant anti-wrinkle activity by inhibiting elastase, downregulating MMPs, and upregulating COL-1 expression. XTRC also demonstrated potent antioxidant activity by reducing ROS production, increasing antioxidant enzymes, and upregulating Nrf2, CAT, SOD, GPx, and GST expressions. Additionally, it showed notable anti- inflammatory activity by reducing chemokines and downregulating NF-κB, TARC, MDC, IL-6, IL-8, and TNF-α expressions, along with good wound healing properties. These findings suggest that XTRC could be a promising formulation for enhancing the bioactivity and stability of phytochemicals, offering a multifaceted approach to combating photoaging.

목차 (Table of Contents)

• I. GENERAL INTRODUCTION. 1
• II. LITERATURE REVIEW 3
• 2.1. Rhus chinensis Mill 3
• 2.2. Phytochemistry of R. chinensis 3
• 2.3. Skin and Photoaging. 8
• 2.4. Molecular Mechanism of Photoaging 10
• 2.5. Nanocarriers Based Drug Delivery 15
• 2.6. Lipid-based Nanocarriers. 17
• 2.7. Transfersomes 17
• 2.8. Xanthan Gum. 22
• III. ENHANCED ANTI-AGING EFFECTS BY SYNERGIC APPLICATION OF Rhuschinensis Mill. FRUIT AND BARK EXTRACT. 24
• 3.1. Abstract. 24
• 3.2. Introduction. 24
• 3.3. Materials and Methods 26
• 3.3.1. Chemicals and Cell Lines 26
• 3.3.2. Plant Materials and Extraction 27
• 3.3.3. Estimation of Total Phenolics and Flavonoid Contents (TPC and TFC) 27
• 3.3.4. Cell Culture 27
• 3.3.5. Estimation of In Vitro Cytotoxicity 28
• 3.3.6. Anti-melanogenic Activity Measurements 28
• 3.3.6.1. Measurement of Mushroom Tyrosinase Inhibition 28
• 3.3.6.2. Determination of Intracellular Tyrosinase Activity in B16F10 Cells 30
• 3.3.6.3. Measurement of Melanin Content 30
• 3.3.7. Anti-wrinkle Activity Measurements 31
• 3.3.7.1. Measurement of Elastase Inhibition 31
• 3.3.7.2. Determination Elastase Activity in HDF Cells 31
• 3.3.8. Anti-oxidant Activity. 32
• 3.3.8.1. DPPH and Reducing Power Assay (RPA) 32
• 3.3.8.2. ROS Assay 32
• 3.3.8.3. Antioxidant Enzyme Assays. 33
• 3.3.9. Anti-inflammatory Activity 34
• 3.3.9.1. Enzyme Linked Immunosorbent Assay (ELISA). 34
• 3.3.10. Quantitative Reverse Transcription-polymerase Chain Reaction (qRT-PCR). 34
• 3.3.11. Statistical Analysis 36
• 3.4. Results and Discussion 37
• 3.4.1. Cytotoxicity of RCF, RCB, and RCFB 37
• 3.4.2. Anti-melanogenic Activity of RCF, RCB, and RCFB 39
• 3.4.2.1. Inhibition of Tyrosinase 39
• 3.4.2.2. Effect of RCF, RCB, and RCFB on Melanin Content. 41
• 3.4.2.3. Effect of RCF, RCB, and RCFB on Expression of Melanogenesis-related Genes 43
• 3.4.3. Anti-wrinkle Activity of RCF, RCB, and RCFB. 45
• 3.4.3.1. Effect of RCF, RCB, and RCFB on Elastase 45
• 3.4.3.2. Effect of RCF, RCB, and RCFB on Expression of Wrinkle-related Genes 47
• 3.4.4. Antioxidant Activity of RCF, RCB, and RCFB. 49
• 3.4.4.1. TPC,TFC, DPPH, and RPA 49
• 3.4.4.2. Effect of RCF, RCB, and RCFB on ROS Generation 51
• 3.4.4.3. Effect of RCF, RCB, and RCFB on Antioxidant Enzymes. 53
• 3.4.4.4. Effect of RCF, RCB, and RCFB on Antioxidant Activity Related Genes. 55
• 3.4.5. Anti-inflammatory Activity of RCF, RCB, and RCFB 57
• 3.4.5.1. Effect of RCF, RCB, and RCFB on Inflammatory Chemokines 57
• 3.4.5.2. Effect of RCF, RCB, and RCFB on Inflammation-Related Genes 59
• 3.5. Conclusion 61
• IV. SYNTHESIS OPTIMIZATION AND CHARACTERIZATION OF TRANSFERSOME GELLOADED WITH Rhus chinensis Mill EXTRACT 62
• 4.1. Abstract. 62
• 4.2. Introduction. 62
• 4.3. Materials and Methods 63
• 4.3.1. Reagents 63
• 4.3.2. Sample Preparation 64
• 4.3.2.1. RCFB Extract Preparation 64
• 4.3.2.2. TRC Preparation 64
• 4.3.2.3. XTRC Preparation 67
• 4.3.3. Characterization and Evaluation of TRC. 69
• 4.3.3.1. Determination of Entrapment Efficiency and Loading Capacity. 69
• 4.3.3.2. Determination of Particle Size and Zeta Potential. 69
• 4.3.3.3. Transmission Electron Microscopy (TEM) Analysis. 69
• 4.3.3.4. Scanning Electron Microscopy (SEM) Analysis. 69
• 4.3.3.5. Atomic Force Microscopy (AFM) Analysis 70
• 4.3.3.6. Fourier Transform Infrared (FTIR) Spectroscopic Analysis 70
• 4.3.4. Determination of pH and Spreading Property. 70
• 4.3.5. Storage and Stability 70
• 4.3.6. Sun Protection Factor( SPF) 71
• 4.4. Results and Discussion 71
• 4.4.1.Optimization of TRC 71
• 4.4.2. Size, Polydispersity Index and Zeta Potential of TRC. 74
• 4.4.3. Morphology of TRC 76
• 4.4.4. FTIR Spectroscopic Analysis of TRC. 78
• 4.4.5. pH and Spreading Property of TRC and XTRC. 80
• 4.4.6. Storage and Stability. 82
• 4.4.7. SPF of TRC and XTRC. 84
• 4.5. Conclusion 86
• V. COSMETIC APPLICATIONS OF TRANSFERSOME GEL LOADED WITH Rhus chinensis Mill. FRUIT AND BARK EXTRACTS 87
• 5.1. Abstract. 87
• 5.2. Introduction. 87
• 5.3. Materials and Methods 88
• 5.3.1. Reagents 88
• 5.3.2. Cell Lines and Culture 89
• 5.3.3. Estimation of Biocompatibility 89
• 5.3.3.1. MTT Assay 89
• 5.3.3.1. Live cell-dead Cell Assay 89
• 5.3.4. Anti-melanogenic Activity Measurements 90
• 5.3.4.1. Determination of Mushroom Tyrosinase Inhibition 90
• 5.3.4.2. Determination of Intracellular Tyrosinase Activity in B16F10 Cells 92
• 5.3.4.3. Determination of Melanin Content 92
• 5.3.5. Anti-wrinkle Activity Measurements. 92
• 5.3.5.1. Determination of Elastase Inhibition. 92
• 5.3.5.2. Determination Elastase Activity in HDF Cells 93
• 5.3.6. Anti-oxidant Activity. 93
• 5.3.6.1. ROS Assay. 93
• 5.3.6.2. Antioxidant Enzyme Assays 94
• 5.3.7. Anti-inflammatory Activity 95
• 5.3.7.1. Enzyme-Linked Immunosorbent Assay (ELISA). 95
• 5.3.8. Quantitative Reverse Transcription-polymerase Chain Reaction (qRT-PCR) 95
• 5.3.9. In Vitro Wound Healing Assay. 97
• 5.3.10. Statistical Analysis 97
• 5.4. Results and Discussion. 97
• 5.4.1. Biocompatibility of TRC and XTRC. 97
• 5.4.1.1. MTT Assay. 97
• 5.4.1.2. Live-dead Cell Assay. 100
• 5.4.2. Anti-melanogenic Activity of TRC and XTRC. 102
• 5.4.2.1. Tyrosinase Inhibition of TRC and XTRC 102
• 5.4.2.2. Effect of TRC and XTRC on Melanin Content. 104
• 5.4.2.3. Effect of TRC and XTRC on Expression of Melanogenesis-related Genes 106
• 5.4.3. Anti-wrinkle Activity of TRC and XTRC 108
• 5.4.3.1. Effect of TRC and XTRC on Elastase 108
• 5.4.3.2. Effect of TRC and XTRC on Expression of Wrinkle-related Genes 110
• 5.4.4. Antioxidant Activity of TRC and XTRC 112
• 5.4.4.1. Effect of RCF, RCB, and RCFB on ROS Generation 112
• 5.4.4.2. Effect of TRC and XTRC on Antioxidant Enzymes. 114
• 5.4.4.3. Effect of TRC and XTRC on Antioxidant Activity-related Genes 116
• 5.4.5. Anti-inflammatory Activity of RCF, RCB, and RCFB 118
• 5.4.5.1. Effect of TRC and XTRC on Inflammatory Chemokines. 118
• 5.4.5.2. Effect of TRC and XTRC on Inflammation-related Gene Expressions 120
• 5.4.6. Wound Healing Activity of TRC and XTRC 122
• 5.5. Conclusion. 124
• VI. GENERAL CONCLUSION 125
• VII. REFERENCES. 128
• 국문 초록. 142
• ACKNOWLEDGEMENTS. 145