Methyl Jasmonate Treated Adventitious Roots of Dendropanax morbifera and Its Phytosome Complex as Potential Functional Products against Inflammation and Lung Cancer

다국어 초록 (Multilingual Abstract)

Phytochemicals play a vital role as antioxidants, exerting diverse physiological impacts that alleviate oxidative stress and provide protection against cellular damage, contributing to cancer prevention avert inflammatory diseases. This study initiated the in vitro cultivation of adventitious roots in Dendropanax morbifera. We compared two extractions solvents-distilled water and 70% EtOH-revealing that 70% EtOH extraction led to higher compound yields based on HPLC analysis. Exploring the impact of various IBA concentrations on biomass and antioxidant activities, our findings demonstrated that the greatest levels of TPC/TFC and antioxidant activities were achieved with an IBA concentration of 2 mg/L. To investigate the effects of different elicitors on biomass and the accumulation of secondary metabolites in D. morbifera adventitious roots (DMAR), we employed methyl jasmonate (MeJA), salicylic acid (SA), chitosan (CHI), and yeast extracts. Our results highlighted MeJA as a substantial enhancer of a particular compound, identified as 3,5-Dicaffeoylquinic acid (3,5- DCQA), through LC-MS and HPLC analysis. Further optimization entailed determining the ideal concentration and exposure time for MeJA-induced production of 3,5-DCQA. Results indicated that when treated with MeJA at a concentration of 40 µM for 36 hours, the maximum level of 3,5-DCQA was achieved. Finally, the enhanced anti-inflammatory and anti-lung cancer activities were investigated. MeJA-DMARE exhibited superior anti-inflammatory activity by inhibiting the production of NO and ROS in LPS-induced RAW 264.7 cells. Moreover, MeJA-DMARE downregulated the mRNA expression of inflammation-related cytokines, including COX-2, TNF- α, iNOS, IL-6, and IL-1β. Additionally, MeJA-DMARE displayed anti-lung cancer activity by promoting ROS production in A549 lung cancer cells, inhibiting their migration at a concentration of 250 µg/mL. Furthermore, it regulated the apoptosis of lung cancer cells through the Bcl-2 and p38 MAPK pathways. This study also aims to create a potential phytosome, a complex of plant extract and phospholipids, to enhance the dermal delivery of MeJA-DMARE and its anti-lung cancer activities. The phytosome complex was created by combining MeJA-DMARE with soy phosphatidylcholine (PC) using 6 different ratios A1, A2, A3, B1, B2, and B3. Initial evaluation based on UV-vis, entrapment efficiency (EE%), and loading content (LC%) indicated that B3 exhibited the highest EE% (79.98 ± 1.45) and LC% (69.17 ± 0.14). The phytosome B3 displayed a spherical morphology as evidenced by transmission electron microscope (TEM) and scanning electron microscope (SEM) analyses. Additionally, the particle size was 210 nm, with a notably low polydispersity index of 0.16. The zeta potential value was recorded as -25.19 mV. In conclusion, the MeJA-DMARE-phytosome offers stable, economically efficient, and environmentally friendly nanoparticles with functional attributes. The study findings indicate that MeJA-DMARE-phytosome retains the same mode of action as MeJA-DMARE but exhibits superior anti-inflammatory and anti-lung cancer properties. These results suggest that MeJA- DMARE-phytosome holds promise as an effective and promising formulation for drug delivery and therapy in lung cancer.

목차 (Table of Contents)

• List of Tables · v
• List of Figures vi
• Abbreviations · viii
• Abstract · x
• I. General introduction · 1
• II. Review of literature · 4
• 2.1. Dendropanax morbifera 4
• 2.1.1. Taxonomy, distribution, and description of Dendropanax species 5
• 2.1.2. Phytochemistry of Dendropanax species · 5
• 2.1.3. Medicinal uses and pharmacological properties of D. morbifera 10
• 2.2. Tissue culture of D. morbifera 17
• 2.3. The effects of elicitors on the accumulation of secondary metabolites 17
• 2.4. Phytosome overview 22
• 2.4.1. Phytosome technology 22
• 2.4.2. Phytosome preparation methods · 24
• 2.4.3. Differences between liposomes and phytosomes 25
• 2.5. Cancer· 27
• 2.6. Inflammation 29
• 2.7. Application of natural phytochemicals in anti-cancer and anti-inflammatory activities · 29
• 2.8. Purpose of studies 30
• III. Tissue culture and phenolic compounds identification in adventitious roots of D.
• morbifera 31
• 3.1. Abstract 31
• 3.2. Introduction 31
• 3.3. Materials and method · 32
• 3.3.1. Materials and chemicals 32
• 3.3.2. Tissue culture of D. morbifera 33
• 3.3.3. The effect of IBA on the secondary metabolites accumulation of D. morbifera · 33
• 3.3.4. Preparation of leaf, stem, root and DMAR extracts (DMLE, DMSE, DMRE and
• DMARE) 34
• 3.3.5. Compounds analysis by HPLC and LC-MS 36
• 3.3.6. Total phenolic content (TPC) measurement 36
• 3.3.7. Total flavonoid content (TFC) measurement · 37
• 3.3.8. Antioxidant activity · 37
• 3.3.9. Statistical analysis 38
• 3.4. Results and discussions 38
• 3.4.1. Adventitious roots induction and the effect of IBA concentration on DMAR · 38
• 3.4.2. Compounds analysis results of DMLE, DMSE, DMRE and DMARE 42
• IV. Anti-lung cancer and anti-inflammatory activities of methyl jasmonate-treated D.
• morbifera adventitious root · 49
• 4.1. Abstract 49
• 4.2. Introduction 49
• 4.3. Materials and methods 51
• 4.3.1. Materials and chemicals 51
• 4.3.2. Elicitors on DMAR for enhancing secondary metabolites 51
• 4.3.3. Antioxidant activity · 52
• 4.3.4. Cell cultures 52
• 4.3.5. Cytotoxicity assay 53
• 4.3.6. Inhibition of nitric oxide (NO) production 53
• 4.3.7. Reactive Oxygen Species (ROS) generation assay · 54
• 4.3.8. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 54
• 4.3.9. Wound healing assay · 55
• 4.3.10. Statistical analysis · 55
• 4.4. Results and discussions 55
• 4.4.1. Effects of elicitors on the compound enhancement of DMARE · 55
• 4.4.2. Effects of concentration and elicitation time of MeJA on 3,5-DCQA production ·· 62
• 4.4.3. Cytotoxicity effect of DMARE and MeJA-DMARE · 66
• 4.4.4. Inhibited NO and ROS generation in RAW 264.7 Cells · 68
• 4.4.5. In vitro ROS generation on lung cancer cells 71
• 4.4.6. Wound healing 73
• 4.4.7. Apoptotic gene expression in lung cancer cells 75
• 4.4.8. The inhibition of the increased levels of inflammation-related cytokines 77
• 4.5. Conclusion · 79
• V. Phytosome for delivering methyl jasmonate-treated adventitious root extracts from
• Dendropanax morbifera to inhibit inflammation and lung cancer 80
• 5.1. Abstract 80
• 5.2. Introduction 81
• 5.3. Materials and methods 82
• 5.3.1. Materials and chemicals 82
• 5.3.2. Synthesis of phytosome by MeJA-DMARE 82
• 5.3.3. Characterization of phytosome 85
• 5.3.4. In vitro stability studies 86
• 5.3.5. Cell cultures 87
• 5.3.6. Cytotoxicity assay 87
• 5.3.7. Inhibition of nitric oxide (NO) production 87
• 5.3.8. Reactive oxygen species (ROS) generation assay 88
• 5.3.9. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) · 88
• 5.3.10. Wound healing assay · 89
• 5.3.11. Statistical analysis · 89
• 5.4. Results and discussions 89
• 5.4.1. Formulation of MeJA-DMARE-phytosome complex · 89
• 5.4.2. Characterization · 91
• 5.4.3. Cytotoxicity MeJA-DMARE and MeJA-DMARE-phytosome 105
• 5.4.4. Inhibited NO and ROS generation in RAW 264.7 Cells 108
• 5.4.5. In vitro ROS generation on lung cancer cells 111
• 5.4.6. Wound healing · 113
• 5.4.7. Apoptotic gene expression in lung cancer cells 115
• 5.4.8. The inhibition of the increased levels of inflammation-related cytokines · 117
• 5.5. Conclusion 119
• VI. General Conclusion 120
• VII. References 121
• 국 문 초 록 · 136
• Acknowledgment 138