Comprehensive Investigation of Sargassum yezoense-Derived Marine Meroterpenoids : Optimized Extraction, Physicochemical Stability, and Pharmacokinetics = 덤불 모자반 유래 해양 메로테르페노이드의 포괄적 연구: 최적화된 추출법, 물리화학적 안정성 및 약동학

다국어 초록 (Multilingual Abstract)

Sargahydroquinoic acid (SHQA) is a meroterpenoid bearing a hydroquinone moiety that is predominantly found in Sargassum species and is known for its potent antioxidant activity. However, its chemical stability and oral bioavailability have not yet been systematically investigated. Therefore, the aims of this study were to (i) optimize SHQA extraction, (ii) elucidate the mechanisms of underlying its chemical degradation in order to develop stabilization strategies, and (iii) evaluate its oral delivery and pharmacokinetic behavior. Extraction of SHQA from Sargassum yezoense was optimized using response surface methodology with a Box-Behnken design to evaluate the effects of extraction temperature, time, and ethanol concentration on SHQA yield. The quadratic model showed good fit (R² = 0.961; F = 13.86, p = 0.005), and ethanol concentration emerged as the most influential factor (p < 0.001). The optimal conditions (52.8 °C, 8.3 h, 74.1% ethanol) yielded an SHQA-maximized extract (SME) 67.8 ± 0.6 mg SHQA/g. SME exhibited strong antioxidant capacity in ABTS, DPPH, and FRAP assays and significantly suppressed H₂O₂-induced reactive oxygen species generation in a zebrafish model (50 µg/mL SME; 1 µg/mL SHQA), thereby establishing a robust extraction platform and confirming antioxidant efficacy in vitro and in vivo. The intrinsic instability of SHQA was elucidated through kinetic and mechanistic analyses of its thermal and oxidative degradation, along with evaluation of acidification and matrix effects. LC-MS/MS profiling and kinetic modeling demonstrated that SHQA undergoes sequential oxidation to sargaquinoic acid (SQA) and subsequent 6π-electrocyclization to sargachromenol (SCM), accompanied by substantial loss of antioxidant capacity. Density functional theory analysis showed that oxidation decreases HOMO energy and alters the electronic structure of the degradation products. Weibull modeling revealed rapid initial degradation (β < 1) under neutral conditions, whereas acidification markedly extended SHQA half-life at 37 °C from 1.28 h (control) to 119.4 h (1% acetic acid) and 3065 h (0.1% HCl). The SME matrix further improved stability (t₁/₂ = 182 h), with an additive stabilizing effect under 1% acetic acid. The gastrointestinal fate and oral pharmacokinetics of SHQA were investigated, and a nanoemulsion-based delivery system was developed to improve its bioavailability. In C57BL/6J mice model (8-12 weeks old, male), oral administration of SHQA in corn oil (20-200 mg/kg) resulted in pronounced gastric retention and progressive decline of SHQA along the small intestine, accompanied by extensive oxidative conversion to SQA and SCM, especially in the ileum. SHQA itself was undetectable in plasma, whereas SQA (and to a lesser extent SCM) appeared systemically with saturable exposure (Cₘₐₓ 0.23-3.18 µg/mL; AUC₀-₂₄ 1.60-54.4 h·µg/mL). Simulated gastric digestion showed substantial degradation of SHQA in the corn oil formulation (53.4 ± 0.3%), while nanoemulsion encapsulation preserved 86.96 ± 0.84% of SHQA and maintained droplet structure. In vivo, nanoemulsion delivery (20 mg/kg) reduced gastric and fecal exposure to SHQA/SQA/SCM and increased plasma SQA levels by approximately 9.8-fold compared with corn oil, indicating improved gastric stability, solubilization, and systemic appearance of its oxidized metabolite. Collectively, this thesis establishes a comprehensive framework for the development of SHQA-based marine functional ingredients and provides a rational basis for designing stable, efficacious SHQA-enriched products, with broader implications for managing redox-sensitive marine meroterpenoids in functional food and nutraceutical applications.

목차 (Table of Contents)

• Chapter 1.Recent Advances in the Extraction and Characterization of Functional Compounds from Sargassum Species 1
• 1. Introduction 1
• 1.1. Research Background and Objectives 1
• 1.2. Marine Algae as Sustainable Sources of Functional Ingredients 3
• 1.3. Ecological and Industrial Significance of Sargassum spp 5
• 2. Functional Compounds in Sargassum: Classification and overview 8
• 2.1. Overview of Functional Metabolites from Sargassum species 8
• 2.2. Classification of Bioactive Compounds 9
• 3. Functional Roles of Sargassum species 19
• 3.1. Polyphenols and Phlorotannins 19
• 3.2. Terpenoids 28
• 4. Extraction Techniques for Bioactive Compounds from Sargassum 43
• 4.1. Conventional Solvent Extraction 43
• 4.1.1. Solvent Selection 44
• 4.2. Optimization Strategies 48
• 5. Bioactive Compounds and Reported Bioactivities of Sargassum yezoense 54
• 5.1. Characteristics 54
• 5.2. Identified Bioactive substances 58
• 5.3. Reported Bioactivities in S. yezoense Extracts 64
• 6. SHQA as a Model Compound: Challenges and Prospects 69
• 6.1. Structural Instability of SHQA 69
• 6.2. Need for Stabilization: Justification for Mechanistic Studies 70
• 6.3. Analytical Challenges and Interpretation of Degradation Kinetics 70
• 7. Enhancing the Bio-accessibility of SHQA through Delivery System 72
• 7.1. Issues in Digestive Stability and Absorption 72
• 7.2. Nanoemulsion, Micelles, and Liposomes: Principles and Examples 74
• 7.3. Application in Marine Polyphenols: Recent Advances 80
• 8. Pharmacokinetic Insights into Hydroquinone-Type Marine Compounds 82
• 8.1. Pharmacokinetics (PK) 82
• 8.2. ADME patterns of Hydroquinone contained compounds 86
• References 89
• Chapter 2. Optimized Extraction of Sargahydroquinoic Acid, Major Bioactive Substance, from Sargassum yezoense Using Response Surface Methodology 99
• Abstract 99
• 1. Introduction 101
• 2. Materials & methods 103
• 2.1. Materials 103
• 2.2. Sample preparation and Extraction 103
• 2.3. Optimization of Extraction Conditions 104
• 2.4. Total Phenolic content (TPC) 105
• 2.5. Total Antioxidant capacity (TAC) by ABTS, DPPH, and FRAP assays 106
• 2.6. Purification Method for SHQA 107
• 2.7. Estimation of Intracellular ROS Generation in Zebrafish Embryos 108
• 2.8. Statistical Analysis 109
• 3. Results 110
• 3.1. RSM analysis of SHQA Extraction 110
• 3.2. Effects of Extraction Temperature, Time, and EtOH Concentration 113
• 3.3. Antioxidant Properties of Extracts from Sargassum yezoense 116
• 3.4. Protective Effect of SME and SHQA against H2O2-Induced Oxidative Stress 118
• 4. Discussion 120
• 5. Conclusions 125
• 6. References 126
• Chapter 3. Kinetics and Mechanisms of Sargahydroquinoic Acid Degradation: Effects of Acidification and Extract Matrix on Stabilization 129
• Abstract 129
• 1. Introduction 130
• 2. Materials & Method 133
• 2.1. Sample Preparation and Thermal Treatment 133
• 2.2. LC-MS/MS Analysis of Meroterpenoids and Metabolites 133
• 2.3. Kinetic Analysis 137
• 2.4. Density Functional Theory 137
• 2.5. Total Antioxidant Capacities by ABTS, DPPH, and FRAP Assays 138
• 2.6. Statistical Analysis 139
• 3. Results and Discussion 140
• 3.1. Thermal Degradation Kinetics of Purified SHQA 140
• 3.2. Formation of SQA and SCM and Antioxidant Loss 143
• 3.3. Structural Conversion of SHQA to SCM via Oxidation and 6π-Electrocyclization 146
• 3.4. Frontier Molecular Orbital and Antioxidant Activity of SHQA, SQA, and SCM 150
• 3.5. Acid-Mediated Stabilization of SHQA 153
• 3.6. Thermal Stability of SHQA in SME 159
• 3.7. Acid-Mediated Stabilization of SHQA in SME 167
• 4. Conclusion 170
• 5. References 171
• Chapter 4. Gastrointestinal Transformation of the Marine Meroterpenoid SHQA and Nanoemulsion-Enabled Improvement in Its Oral Bioavailability 174
• 1. Introduction 176
• 2. Materials & Methods 179
• 2.1. Materials 179
• 2.2. Sample preparation 179
• 2.3. Simulated digestion system 179
• 2.4. Animals and Ethical Approval 180
• 2.5. Pharmacokinetic Study 180
• 2.6. Quantification of SHQA, SCM and SQA 181
• 2.7. Particle Size Analysis and Morphological Evaluation of Nanoemulsion 182
• 2.8. Molecular Docking 183
• 2.9. Statistical Analysis 183
• 3. Results and Discussion 183
• 3.1. Swiss Prediction of SHQA 183
• 3.2. Absorption of SHQA after Oral Administration 186
• 3.3. Distribution and Metabolism of SHQA After oral Administration 196
• 3.4. Excretion of SHQA after Oral Administration 204
• 3.5. Formulation of SHQA Containing Nanoemulsion 209
• 3.6. Stability of SHNM under the Gastric Conditions 212
• 3.7. Effects of Nanoemulsion on Tissue Distribution and Systemic Exposure of SHQA 217
• 4. Conclusion 227
• 5. Reference 228