Protective effects of the extract of Lithospermum erythrorhizon and active compounds on allergic and inflammatory diseases = 지치 추출물과 유효성분의 알러지 및 염증성 질환에 대한 보호효과 연구

다국어 초록 (Multilingual Abstract)

General Abstract.
This dissertation investigates the protective effects of the extract of Lithospermum erythrorhizon (LE) and its active compounds on allergic and inflammatory diseases, focusing on allergic rhinitis and uveitis. LE is a medicinal plant that has been used in traditional medicine for various purposes, such as anti-inflammatory, antipyretic, and anti-allergic. The dissertation aims to identify the various active components of LE that mediate its activities and is divided into two chapters. Each chapter explores the protective effects of LE and active compounds in models of allergic and inflammatory diseases.
Chapter 1 examines the antiallergic effects of LE and N,N`-dicoumaroylspermidine, a novel compound isolated from LE, on mast cells and ovalbumin (OVA)-induced allergic rhinitis in mice. The chapter demonstrates that LE and N,N`-dicoumaroylspermidine can effectively suppress the allergic response by inhibiting mast cell degranulation, reducing the production of Th2 cytokines and OVA-specific immunoglobulins, and alleviating nasal symptoms and inflammation.
Chapter 2 investigates the anti-inflammatory effects of LE and three of its compounds, shikonin, acetylshikonin, and β, β-dimethylacryloylshikonin, on endotoxin-induced uveitis (EIU) in rats and mice. The chapter shows that LE and its compounds can significantly reduce the severity of uveitis by decreasing the clinical inflammatory score, retinal thickness, protein concentration, prostaglandin E2 levels, and cellular infiltration in the aqueous humor. The chapter also reveals that LE and its compounds can inhibit the expression of pro-inflammatory cytokines and the activation of NF-κB/AP-1 and IRF signaling pathways in vitro and in vivo.
The dissertation concludes that LE and its active compounds have potent protective effects on inflammatory and allergic diseases, especially allergic rhinitis and uveitis, and suggests that they could be potential candidates for developing new plant-based therapies for these conditions. The dissertation also provides novel insights into the chemical diversity and pharmacological mechanisms of LE and its constituents.

Chapter1 Abstract.
In East Asia, the dried root of LE has a long history of use as an anti-inflammatory, antipyretic, detoxifying, and anti-allergic agent. Building on our previous findings that LE offers protective effects against allergic rhinitis, this study delves deeper to identify and characterize the specific antiallergic active components within an ethanolic extract of LE roots. To achieve this, we employed an ovalbumin (OVA)-induced allergic rhinitis mouse model to evaluate the antiallergic effects of LE reflux ethanol extracts. A comparative analysis was conducted between the chemical compounds extracted using both hot reflux and cold extraction methods to ascertain the most effective extraction technique. Through chromatographic separation, our research successfully identified two novel anthraquinones, named erythrin A and B, and a newly discovered compound unique to the Lithospermum genus, N,N`-dicoumaroylspermidine, along with nineteen other recognized compounds. The structural elucidation of these compounds was achieved through comprehensive single (1D) and 2D nuclear magnetic resonance (NMR) spectroscopic data analysis, supported by high-resolution mass spectrometry. Of the compounds identified, N,N`-dicoumaroylspermidine emerged as a standout, demonstrating a strong inhibition of β-hexosaminidase release, and significantly reducing the production of interleukins IL-3, IL-4, and IL-13 in IgE-sensitized and BSA-stimulated RBL-2H3 cells. Furthermore, using the OVA-induced allergic rhinitis mouse model, N,N-dicoumaroylspermidine showed a notable reduction in serum OVA-specific IgE levels and a decrease in the number of inflammatory cells in nasal lavage fluid. These findings underscore the potential of N,N-dicoumaroylspermidine, isolated from LE, as an effective antiallergic agent, particularly for the management of allergic rhinitis. This study not only sheds light on the antiallergic properties of LE but also opens avenues for further research into its therapeutic applications in allergic conditions.

Chapter2 Abstrat.
Uveitis is an inflammatory eye condition that threatens vision, and effective anti-inflammatory treatments with minimal side effects are necessary to treat uveitis. This study aimed to investigate the effects of LE against endotoxin-induced uveitis in rat and mouse models. Endotoxin-induced uveitis models of rats and mice were used to evaluate the effects of LE treatment. Clinical inflammation scores and retinal thickness were assessed in the extract of LE rats. Histopathological examination revealed inflammatory cell infiltration into the ciliary body. Protein concentration, cellular infiltration, and prostaglandin-E2 levels were measured in the aqueous humor of the extract of LE-treated rats. Protective effects of LE on the anterior segment of the eye were examined in mice with endotoxin-induced uveitis. Additionally, we investigated the effect of LE the expression of pro-inflammatory cytokines in lipopolysaccharide-stimulated THP1 human macrophages and examined the involvement of nuclear factor kappaB (NF-κB)/activator protein 1 and interferon regulatory factor signaling pathways. Furthermore, three components of LE were identified and assessed for their inhibitory effects on LPS-induced inflammation in RAW264.7 macrophage cells. Treatment of the extract of LE significantly reduced clinical inflammation scores and retinal thickening in rats with endotoxin-induced uveitis. Histopathological examination revealed decreased inflammatory cell infiltration into the ciliary body. The extract of LE effectively reduced the protein concentration, cellular infiltration, and PG-E2 levels in the aqueous humor of rats with endotoxin-induced uveitis. In mice with endotoxin-induced uveitis, the extract of LE demonstrated a protective effect on the anterior segment of the eye by reducing inflammation and retinal thickening. The extract of LE suppressed the expression of pro-inflammatory cytokines in lipopolysaccharide-induced inflammation in THP1 human macrophages, by modulating NF-κB/activator protein 1 and interferon regulatory factor signaling pathways. Moreover, shikonin, acetylshikonin, and β, β-dimethylacryloylshikonin showed dose-dependent inhibition of nitric oxide, NF-κB and interleukin-6 production in RAW264.7 macrophage cells. The extract of LE is a potential therapeutic agent for uveitis management. Administration of the extract of LE led to reduced inflammation, retinal thickening, and inflammatory cell infiltration in rat and mouse models of uveitis.

목차 (Table of Contents)

• List of Figures 5
• Abbreviations 9
• Abstract 10
• Chapter 1. 12
• Ⅰ. Abstract 13
• Ⅱ. Introduction 15
• Ⅲ. Methods and Material 26
• 3-1. Plant Material and Extraction 26
• 3-2. Chemicals and Apparatus 27
• 3-3. Cell Culture and Viability Assay 28
• 3-4. β-Hexosaminidase Assay 29
• 3-5. Real-Time Quantitative PCR 30
• 3-6. Measurement of NF-κB/AP-1 and IRF Activity 31
• 3-7. OVA-Induced Allergic Rhinitis Mouse Model 32
• 3-8. Collection and Analysis of Serum and NALF 33
• 3-9. Histopathological Evaluation of Nasal Mucosa 34
• 3-10. Statistical Analysis 35
• Ⅳ. Results 36
• 4-1. HPLC Analysis of Extracts of LE 36
• 4-2. LE Significantly Ameliorated Nasal Allergy Symptoms and
• Reduced NALF cytokine Levels in Mice with OVA-Induced
• AR 39
• 4-3. Effects of LE Extract on Levels of Th2 Cytokines in Serum and the Nasal Lavage Fluid of OVA-Induced Allergic Rhinitis Mice 42
• 4-4. LE Significantly Suppressed Allergic Responses by Modulating OVA-Specific Immunoglobulins in the Serum of OVA-Induced AR Mice 45
• 4-5. LE Inhibited IgE-Stimulated Degranulation and Histamine Release by RBL-2H3 Cells 48
• 4-6. LE Ethanol Extract Causes Histopathological Changes in
• Tissues from OVA-Induced Allergic Rhinitis Mice Model 50
• 4-7. The Effects of 11 Constituents on Viability of THP1 Macrophages and RBL-2H3 Basophils 54
• 4-8. Assessment of NF-kB, AP1, and IRF Activation 56
• 4-9. Antiallergic Effects of N,N`-dicoumaroylspermidine on Mast Cells (RBL-2H3) 58
• 4-10. Effects of N,N`-dicoumaroylspermidine on Serum Levels of OVA-Specific IgE and Inflammatory Cells in the NALF of OVA-Induced Allergic Rhinitis Mice Model 61
• Ⅴ. Discussion 65
• Chapter 2. 72
• Ⅰ. Abstract 73
• Ⅱ. Introduction 75
• Ⅲ. Methods and Material 85
• 3-1. Chemicals and reagent 85
• 3-2. Plant materials 87
• 3-3. Animals and induction of uveitis 88
• 3-4. Clinical inflammatory score 90
• 3-5. Histological examination of infiltrating cells 91
• 3-6. Cell counts, protein concentration, and PG-E2 levels
• in the aqueous humor 92
• 3-7. Cell culture 93
• 3-8. Cell viability 94
• 3-9. Enzyme-linked immunosorbent assay (ELISA) 95
• 3-10. Quantification of NF-κB/AP-1, IRF and TLR4/NF-κB
• activity 96
• 3-11. Nitric oxide (NO) assay 97
• 3-12. Statistical Analysis 98
• Ⅳ. Results 99
• 4-1. Effects of LE on the clinical inflammation score and
• retinal thickness 99
• 4-2. Effects of LE on histology 102
• 4-3. Effects of LE on the protein concentration and PG-E2
• levels in the AqH 105
• 4-4. Effects of LE on the anterior segment of the eye in mice
• with EIU 108
• 4-5. LE suppresses pro-inflammatory cytokine expression
• via the NFkB/AP1 and IRF signaling pathways 112
• 4-6. Three compounds isolated from LE inhibited inflammatory
• responses 115
• 4-7. Inhibition of NFκB/AP1 and IRF pathways by shikonin,
• acetylshikonin, and β, β- dimethylacryloylshikonin 120
• Ⅴ. Discussion 122
• Ⅵ. Reference 130
• Ⅶ. Abstract in Korean 146