Phytochemical Characterization and Nanoformulation Strategies for Enhanced Delivery of Parsley (Petroselinum crispum) Extracts

다국어 초록 (Multilingual Abstract)

Parsley (Petroselinum crispum) is a valuable source of bioactive compounds, yet the hydrophobic nature of its constituents limits their effective application. This dissertation focuses on the valorization of parsley n-hexane fraction, rich in kaempferol, through the development of tailored delivery systems to enhance its stability, solubility, and functional use. In the first study, parsley extracts were evaluated for their chemical composition and antioxidant activity. Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) profiling confirmed the presence of kaempferol in the n-hexane fraction, which also demonstrated potent radical scavenging activity in the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. To improve its delivery, a self-nanoemulsifying drug delivery system (SNEDDS) was initially developed using coconut oil, selected for its dual applicability in both oral and topical formulations. A D-optimal mixture design was employed to optimize droplet size, polydispersity index (PDI < 0.4), and clarity (>80% transmittance). This nanoemulsion was further explored as a topical system, where in vivo studies indicated enhanced hair growth activity. To adapt the formulation for oral administration in a food-grade context, the SNEDDS was reformulated using fish oil and solidified into self-nanoemulsifying granules (SNEGs) using pullulan. The resulting granules exhibited excellent flowability, rapid reconstitution (<2 minutes), and sustained release behavior in simulated gastrointestinal conditions. Collectively, this work offers an integrated approach that combines plant extract characterization with advanced delivery technologies. By leveraging SNEDDS and SNEG platforms, this research provides a functional solution for delivering hydrophobic bioactives like parsley n-hexane fraction in both pharmaceutical and nutraceutical applications.

국문 초록 (Abstract)

파슬리(Petroselinum crispum)는 다양한 생리활성 화합물을 함유한 유용한 식물 자원으로 알려져 있으나, 그 구성 성분의 소수성 특성으로 인해 활용 효율이 제한적이다. 본 학위논문은 켐페롤(kaempferol)이 풍부한 파슬리 n-헥산 분획을 고부가가치화하기 위해, 그 안정성·용해도·기능적 활용성을 향상시킬 수 있는 맞춤형 전달 시스템의 개발에 초점을 두었다. 첫 번째 연구에서는 파슬리 추출물의 화학적 조성과 항산화 활성을 평가하였다. LC-MS/MS 분석 결과, n-헥산 분획에서 켐페롤이 확인되었으며, DPPH 라디칼 소거 실험을 통해 우수한 항산화 활성이 입증되었다. 이를 효율적으로 전달하기 위해, 경구 및 피부 적용이 모두 가능한 코코넛오일을 기반으로 한 자가 나노유화 약물 전달 시스템(Self-Nanoemulsifying Drug Delivery System, SNEDDS)을 개발하였다. 이후 D-최적 혼합 설계를 적용하여 입자 크기, 분산지수(PDI < 0.4), 투명도(>80%)를 최적화하였다. 개발된 나노에멀전은 피부 도포용 시스템으로도 적용 가능성을 검토하였으며, in vivo 실험 결과 모발 성장 촉진 효과가 향상됨을 확인하였다.
식품 등급 기반의 경구 제형으로의 응용을 위해, SNEDDS를 어유(fish oil)로 재구성하고 풀루란(pullulan)을 이용하여 고형 자가 나노유화 과립(Self-Nanoemulsifying Granules, SNEGs)으로 전환하였다. 제조된 과립은 우수한 유동성, 2분 이내의 빠른 재분산성, 모의 위장관 조건에서의 지속 방출 특성을 나타내었다. 종합적으로, 본 연구는 식물 추출물의 화학적 특성 규명과 고기능 전달 기술을 융합한 통합적 접근법을 제시한다. SNEDDS 및 SNEG 플랫폼을 활용함으로써, 파슬리 n-헥산 분획과 같은 소수성 생리활성 물질의 약학적 및 건강기능식품 응용 가능성을 확대할 수 있는 기능적 전달 전략을 제안하였다.

목차 (Table of Contents)

• Chapter I. Overall Introduction 1
• Overall Introduction 2
• Chapter II. Metabolite Contents Identification and In Vitro Antioxidant Activity of N-Hexane Fraction and Ethanolic Extract from Parsley (Petroselinum crispum) Cultivated in South Korea 5
• 2.1. Introduction 6
• 2.2. Material and method 9
• 2.2.1. Plant material 9
• 2.2.2. Extraction method 9
• 2.2.3. Fractionation method 9
• 2.2.4. Antioxidant activity test (DPPH assay) 10
• 2.2.5. LCMS/MS 11
• 2.3. Results and discussion 12
• 2.3.1. Extraction and fractionation 12
• 2.3.2. DPPH assay 15
• 2.3.3. Metabolite contents identification 19
• 2.4. Conclusion 29
• Chapter III. Design, Optimization, and Evaluation of a Self-Nanoemulsifying Drug Delivery System (SNEDDS) for Oral Delivery of Parsley N-Hexane Fraction Utilizing Coconut Oil as the Oil Phase 30
• 3.1. Introduction 31
• 3.2. Material and method 35
• 3.2.1. Plant material 35
• 3.2.2. Ultrasound-assisted extraction 35
• 3.2.3. Solvent-solvent partitioning 35
• 3.2.4. Fraction solubility study 36
• 3.2.5. Experimental design and optimization of SNEDDS 36
• 3.2.6. Parsley SNEDDS formulation 37
• 3.2.7. Droplet size analysis and polydispersity index 37
• 3.2.8. Zeta potential 37
• 3.2.9. Clarity test 37
• 3.2.10. Robustness to dilution 38
• 3.2.11. Emulsification test 38
• 3.2.12. Cryo-TEM analysis 38
• 3.2.13. In vitro drug release study 38
• 3.2.14. Centrifugation test 39
• 3.2.15. Heating-cooling cycle test 39
• 3.2.16. Freeze-thaw cycle test 39
• 3.2.17. Endurance test 40
• 3.3. Results and discussion 41
• 3.3.1. Fraction preparation and solubility test 41
• 3.3.2. Experimental design and optimization of SNEDDS 41
• 3.3.3. Parsley SNEDDS formulation and evaluation 47
• 3.3.4. Stability Study 56
• 3.4. Conclusion 60
• Chapter IV. Parsley (Petroselinum crispum) Fraction-Loaded Nanoemulsion Using Coconut Oil: A Novel Approach for Hair Growth Promotion 61
• 4.1. Introduction 62
• 4.2. Material and method 65
• 4.2.1. Plant material 65
• 4.2.2. Parsley extraction and fractionation 65
• 4.2.3. Preparation of parsley nanoemulsion 66
• 4.2.4. Nanoemulsion characterization 66
• 4.2.5. Antioxidant activity 66
• 4.2.6. In vivo hair growth study 67
• 4.3. Results and discussion 68
• 4.3.1. Parsley nanoemulsion preparation 68
• 4.3.2. Nanoemulsion characteristic evaluation 68
• 4.3.3. Antioxidant activity 71
• 4.3.4. In vivo hair growth study 73
• 4.4. Conclusion 77
• Chapter V. Pullulan-Based Self-Nanoemulsifying Granules: A Functional Carbohydrate Matrix for Stabilizing and Modulating the Release of Hydrophobic Extracts 78
• 5.1. Introduction 79
• 5.2. Material and method 83
• 5.2.1. Material 83
• 5.2.2. Preparation and optimization of liquid SNEDDS 83
• 5.2.3. Droplet size and polydispersity index (PDI) 85
• 5.2.4. Zeta potential 85
• 5.2.5. Clarity test 85
• 5.2.6. Emulsification test 86
• 5.2.7. Transmission electron microscopy (TEM) analysis 86
• 5.2.8. Thermodynamic stability study 86
• 5.2.9. Robustness to dilution 86
• 5.2.10. Fabrication of pullulan-based self-nanoemulsifying granules (SNEGs) 87
• 5.2.11. Appearance and surface morphology. 87
• 5.2.12. Fourier transform infrared spectroscopy (FTIR-ATR) 88
• 5.2.13. X-ray diffraction (XRD) analysis 88
• 5.2.14. Differential scanning calorimetry (DSC) analysis 88
• 5.2.15. Evaluation of reconstituted nanoemulsion 89
• 5.2.16. Storage stability study 89
• 5.2.17. In vitro release study 89
• 5.2.18. Statistical analysis 90
• 5.3. Results and discussion 91
• 5.3.1. Optimization and preparation of SNEDDS 91
• 5.3.2. Characterization of SNEDDS and PSNEDDS 95
• 5.3.3. Stability of SNEDDS and PSNEDDS 98
• 5.3.4. Fabrication and characterization of pullulan-based SNEGs 101
• 5.3.5. Evaluation of reconstituted nanoemulsions 107
• 5.3.6. Storage stability of SNEGs 110
• 5.3.7. In vitro drug release study 112
• 5.3.8. Functional and comparative evaluation of pullulan-based SNEGs 115
• 5.4. Conclusion 119