Biotechnological Strategies for Enhancing Secondary Metabolite Production and Assessing Biological Activities in Cell Suspension Cultures of Lavandula Species = Lavandula 세포배양을 통한 기능성 2차대사산물 생산 증진 및 생물활성 평가

다국어 초록 (Multilingual Abstract)

Plants of the genus Lavandula are widely recognized for their aromatic and medicinal properties, largely attributed to phenolic compounds such as rosmarinic acid (RA), which exhibits strong antioxidant and anti-inflammatory activities. However, conventional field cultivation often results in variable metabolite yields and limited reproducibility, highlighting the need for a controllable and sustainable production system. To address this limitation, this study established cell suspension cultures of Lavandula angustifolia (lavender) and Lavandula × intermedia (lavandin) and investigated the effects methyl jasmonate (MJ) elicitation on secondary metabolite biosynthesis and biological functions. In L. angustifolia cell cultures, MJ treatment markedly enhanced RA accumulation through coordinated upregulation of phenylpropanoid pathway genes. The MJ-treated cell extract (LC-MJ) displayed multifunctional bioactivities in mammalian cell models, including antioxidant, anti-melanogenic, collagen-promoting, and Ultraviolet B (UVB)-protective effects, confirming their potential for cosmeceutical applications. Building on these results, lavandin cell cultures exhibiting higher metabolic vigor were further analyzed. MJ elicitation stimulated RA biosynthesis in lavandin cells, and the resulting extract (LIC-MJ) exhibited strong anti-inflammatory activity by suppressing nitric oxide and cytokine production in macrophages. Similar effects were also observed in extracellular nanovesicles (LIC-NVs) derived from the same cultures, indicating that the bioactive potential of lavandin cells can be reflected in both intracellular and extracellular fractions. The nanovesicles displayed a characteristic bilayered morphology and stable negative charge, and their biological activity suggests that elicitor-induced metabolic reprogramming of plant cells can influence the functional properties of secreted vesicles. Collectively, these findings demonstrate that MJ-elicited Lavandula cell cultures represent a sustainable and scalable biotechnological platform for producing high-value bioactive metabolites and nanovesicles. This study provides new insight into how metabolic modulation at the cellular level can be translated into enhanced extracellular functionality, bridging plant cell biotechnology with natural nanovesicle research and opening new opportunities for developing next-generation antioxidant and anti-inflammatory materials.

국문 초록 (Abstract)

라벤더속(Lavandula) 식물은 특유의 향기와 약리학적 효능으로 널리 알려져 있으며, 이러한 생리활성은 주로 항산화 및 항염 효과가 우수한 로즈마린산(rosmarinic acid, RA)과 같은 페놀 화합물에 기인한다. 그러나 포장 재배에 의한 기존 생산 방식은 대사산물 함량이 불균일하고 재현성이 낮다는 한계를 지니므로, 대사 조절이 가능한 지속가능한 생산 시스템의 확립이 필요하다. 본 연구에서는 Lavandula angustifolia(라벤더)와 Lavandula × intermedia(라반딘)의 세포 배양 기술을 확립하고, 메틸자스모네이트(methyl jasmonate, MJ) 처리에 따른 2 차 대사산물 생합성과 생물학적 활성을 평가하였다. 라벤더 세포 추출물은 MJ 처리에 의해 페닐프로파노이드 경로 관련 유전자들의 발현이 조절되면서 RA 축적이 현저히 증가하였으며, MJ 처리 세포 추출물(LC-MJ)은 항산화, 미백, 콜라겐 합성 촉진, 자외선(UVB) 보호 등 다중 생리활성을 나타내어 기능성 화장품 원료로서의 가능성을 확인하였다. 이러한 결과를 바탕으로 더 높은 대사 활성을 보이는 라반딘 세포 추출물에 대해서도 후속 연구를 수행하였다. 라반딘 세포 배양에서의 MJ 처리는 RA 생합성을 촉진하였으며, 얻어진 추출물(LIC-MJ)은 대식세포에서 산화질소 및 염증성 사이토카인 생성을 억제함으로써 강력한 항염증 활성을 보였다. 또한 동일한 배양계에서 분비된 세포 외 나노소포체(LIC-NVs)에서도 유사한 억제 효과가 관찰되어, 라반딘 세포의 생리활성이 세포 내 성분뿐 아니라 분비 소포에도 반영됨을 확인하였다. 이들 나노소포체는 이중 지질막 구조와 안정적인 음전하를 나타냈으며, 이러한 생물학적 특성은 활성유도제(elicitor)에 의해 유도된 식물 세포의 대사 재프로그래밍이 분비 소포의 기능적 특성에까지 영향을 미칠 수 있음을 시사한다. 종합적으로, MJ 처리된 라벤더속 세포 추출물은 고부가가치 생리활성 물질과 나노소포체를 생산할 수 있는 지속가능하고 확장 가능한 식물 세포 공장형 플랫폼으로서의 가능성을 제시하였다. 본 연구는 식물 세포 수준의
대사 조절이 세포 외 기능성 향상으로 이어질 수 있음을 규명함으로써, 식물 세포공학과 천연 나노소포체 연구를 융합하는 새로운 방향성을 제시하였다.

목차 (Table of Contents)

• Abstract 1
• Chapter 1. General introduction 4
• 1. Background and significance 4
• 1.1. Plant cell culture as a biotechnological platform 6
• 1.2. Rosmarinic acid: a multifunctional phenolic compound 11
• 1.3. Expanding potential through elicitation and nanovesicles 16
• 2. Research objectives and scope 19
• 3. References 20
• Chapter 2. Elicitor-mediated enhancement of rosmarinic acid biosynthesis in lavender (Lavandula angustifolia) cell suspension cultures 26
• Abstract 26
• 1. Introduction 28
• 2. Materials and methods 30
• 2.1. Plant material and callus induction 30
• 2.2. Cell suspension cultures and growth analysis 30
• 2.3. Evans blue staining of lavender cells 31
• 2.4. Elicitor treatment and sample collection 31
• 2.5. DPPH radical scavenging assay 31
• 2.6. RA biosynthesis related gene expression analysis 32
• 2.7. Scale-up culture and cell extraction 32
• 2.8. Quantification of RA by High-Performance Liquid Chromatography (HPLC) 33
• 2.9. Metabolite profiling by gas chromatographymass spectrometry (GC-MS) 34
• 2.10. Statistical analysis 34
• 3. Results 36
• 3.1. Callus induction, establishment, and growth characteristics of suspension cultures 36
• 3.1.1. Stem explants form friable callus enabling the establishment of cell suspension cultures 36
• 3.1.2. Cell suspension cultures show biomass accumulation 36
• 3.2. Metabolite production in L. angustifolia suspension cultures 39
• 3.2.1. MJ treatment enhances RA accumulation compared with other elicitors 39
• 3.2.2. MJ-treated extracts exhibit the strongest antioxidant activity among elicitor treatments 42
• 3.2.3. RA production peaks under 100 M MJ treatment for 3 days 44
• 3.2.4. MJ treatment upregulates RA biosynthesis-related genes 47
• 3.2.5. MJ treatment alters metabolite composition and enriches DDMP and HMB 49
• 4. Discussion 53
• 5. References 60
• Chapter 3. In vitro evaluation of multifunctional skin-related bioactivities of Lavandula angustifolia cell extracts 66
• Abstract 66
• 1. Introduction 67
• 2. Materials and methods 69
• 2.1. Free radical scavenging activity assays 69
• 2.1.1. DPPH radical scavenging activity 69
• 2.1.2. ABTS radical scavenging activity 69
• 2.2. Skin-related functional assays 70
• 2.2.1. Mammalian cell-based viability assays 70
• 2.2.1.1. MTT assay (B16F10 melanoma cells) 70
• 2.2.1.2. WST-1 assay (CCD-986sk human fibroblasts) 71
• 2.2.2. Melanin inhibition assay 71
• 2.2.3. Collagen synthesis assay 72
• 2.3. Photoprotective assays under UVB irradiation 72
• 2.3.1. NIH-3T3 cell viability (CCK-8 assay) 72
• 2.3.2. Cell cycle analysis 73
• 2.3.3. Apoptosis assay (Annexin V/PI) 74
• 2.3.4. Measurement of intracellular ROS levels in NIH-3T3 cells 75
• 2.3.5. Assessment of mitochondrial membrane potential (MMP), mitochondria mass 75
• 2.4. Anti-inflammatory assays in LPS-stimulated RAW 264.7 macrophages 76
• 2.4.1. RAW264.7 cell viability (CCK-8 assay) 77
• 2.4.2. Measurement of intracellular ROS levels in RAW 264.7 cells 77
• 2.4.3. Nitric oxide (NO) determination 78
• 2.4.4. Cytokines quantification 79
• 2.4.5. Western blot analysis 79
• 2.5. Statistical analysis 81
• 3. Results 82
• 3.1. Skin-related functional activities of L. angustifolia cell extracts from a 1-ton bioreactor 82
• 3.1.1. Strong antioxidant activity is observed in lavender cell extracts using DPPH and ABTS assays 82
• 3.1.2. Cell extracts strongly inhibit melanin synthesis in B16F10 melanoma cells 84
• 3.1.3. Cell extracts promote procollagen synthesis in CCD-986sk fibroblasts 86
• 3.2. Photoprotective effects of L. angustifolia cell extracts against UVB-induced oxidative damage 88
• 3.2.1. Treatment with lavender cell extracts protects NIH-3T3 fibroblasts from UVB-induced cytotoxicity 88
• 3.2.2. UVB-induced cell cycle arrest and apoptosis are reduced by LC-MJ treatment 90
• 3.2.3. LC-MJ treatment suppresses intracellular ROS accumulation and prevents mitochondrial dysfunction under UVB exposure 93
• 3.3. Anti-inflammatory activities of L. angustifolia cell extracts in LPS-stimulated RAW 264.7 macrophages 97
• 3.3.1. LC-MJ treatment maintains viability and reduces NO and ROS production in LPS-stimulated RAW 264.7 macrophages 97
• 3.3.2. Secretion of pro-inflammatory cytokines is suppressed by LC-MJ exposure 99
• 3.3.3. LC-MJ suppress pro-inflammatory mediators by inhibiting MAPK and NF-κB signaling pathways 102
• 4. Discussion 104
• 5. References 107
• Chapter 4. Anti-inflammatory activities of lavandin (Lavandula x intermedia) cell extracts and cell culture-derived nanovesicles elicited by methyl jasmonate 112
• Abstract 112
• 1. Introduction 114
• 2. Materials and methods 117
• 2.1. Plant materials and callus induction 117
• 2.2. Cell suspension cultures and cell growth analysis 117
• 2.3. Elicitation and sample collection 117
• 2.4. HPLC analysis of RA 118
• 2.5. Preparation of cell extracts (LIC-CK and LIC-MJ) 118
• 2.6. Isolation of lavandin cell culture-derived nanovesicles (LIC-NVs) 119
• 2.7. Characterization of LIC-NVs 120
• 2.8. Fluorescent labeling and cellular uptake analysis of LIC-NVs 120
• 2.9. Mammalian cell culture and treatments 121
• 2.10. Quantitative real-time RCR (RT-qPCR) analysis 122
• 2.10.1. Expression of RA biosynthesis-related genes in lavandin cells 122
• 2.10.2. Expression of inflammation-related genes in RAW 264.7 macrophages 122
• 2.11. Cell viability assay 123
• 2.12. Measurement of NO production 123
• 2.13. Measurement of pro-inflammatory cytokines (ELISA) 124
• 2.14. Protein isolation and Western blotting analysis 124
• 2.15. Statistical analysis 124
• 3. Results 126
• 3.1. Stem explants form friable callus, and established suspension cultures exhibit characteristic growth and biomass accumulation 126
• 3.2. Elicitor-mediated regulation of RA biosynthesis in L. intermedia cell suspension cultures 128
• 3.2.1. MJ serves as the most effective elicitor, enhancing RA biosynthesis in L. intermedia cell suspension cultures 128
• 3.2.2. MJ coordinately activates genes involved in RA biosynthesis in L. intermedia cell suspension cultures 130
• 3.3. L. intermedia cell cultures produce and release extracellular nanovesicles with defined properties 133
• 3.4. Anti-inflammatory effects and mechanisms of lavandin cell extracts in RAW 264.7 macrophages 135
• 3.4.1. LIC-MJ attenuates LPS-induced inflammatory responses in RAW 264.7 macrophages 135
• 3.4.2. Lavandin cell extracts suppress NF-κB signaling while activating the AMPK/Nrf2/HO-1 antioxidant pathway 139
• 3.5. LIC-NVs suppress LPS-induced inflammatory responses in RAW 264.7 macrophages 147
• 3.6. LIC-NVs are efficiently internalized in RAW 264.7 macrophages 151
• 4. Discussion 154
• 5. References 163
• Abstract (in Korean) 169
• Acknowledgements 171