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研究生: 葉科宏
Yeh, Ke-Hung
論文名稱: 評估不同陳化程度之黑檸檬水萃物抗氧化活性與其對小鼠巨噬細胞之抗發炎影響
Evaluation of the antioxidant properties of aqueous extract of black lemon (Citrus limon L. Brum. f.) at different aged periods and the anti-inflammatory effect in murine macrophages
指導教授: 朱永麟
Chu, Yung-Lin
學位類別: 碩士
Master
系所名稱: 國際學院 - 食品科學國際碩士學位學程
International Master's Degree Program in Food Science
論文出版年: 2023
畢業學年度: 111
語文別: 英文
論文頁數: 109
中文關鍵詞: 陳化抗氧化能力黑檸檬抗發炎反應梅納反映
外文關鍵詞: Aging process, black lemon, antioxidant, anti-inflammatory, Maillard reaction
DOI URL: http://doi.org/10.6346/NPUST202300037
相關次數: 點閱:44下載:0
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  • 檸檬 (Citrus limon L. Burm. f.) 是台灣的一種重要的作物,其含有大量的類黃酮,已有多項研究證明能對於健康帶來益處,然而,在產季期間巨大的產量常造成市場滯銷的問題,因此,開發檸檬相關的多樣化產品是十分必要的。黑檸檬,又稱作陳年檸檬,通常是由檸檬製成的一種柑橘類乾果,經過微熱加工,直到呈現出黑褐色的外觀,並經過乾燥後保存。一般來說,炎症 (發炎) 是身體的免疫系統對刺激物的反應。腸道的炎症與一氧化氮 (NO) 的合成有著高度的相關性,這是由於誘導性一氧化氮合成酶 (iNOS) 上調的結果所導致。本研究將透過福林試劑 (FolinCiocalteu) 比色法和氯化鋁比色法分別測定黑檸檬水萃物 (ABLE) 中的總酚含量和總黃酮含量,並通過鐵還原抗氧化能力 (FRAP) 來測定ABLE 的抗氧化能力,除此之外也測定了陳化過程中其總含糖量的變化。利用 Griess 反應測量由脂多醣 (LPS) 誘導的小鼠巨噬細胞 (RAW264.7) 釋放到培養基中的亞硝酸鹽之含量,在比較不同濃度之 ABLE 對於其抑制效果。對於抗炎之研究,通過西方墨點法(Western blotting) 進行對iNOS、COX-2、IL-6 以及 TNF-α 等促炎介質之評估。綜上所述,研究結果表明,ABLE 具有較高的總酚及總類黃酮含量,以及較佳的鐵環原能力,同時,在分析促炎因子蛋白的方面,也呈現出不錯的抑制效果,尤其是對 iNOS 蛋白的抑制效果有特別突出的表現,由此可知 ABLE 同時具有傑出的抗氧化以及抗發炎能力,未來能夠被應用於開發機能性食品原料之潛力。

    Lemon (Citrus limon L. Brum. f.) is an important crop in Taiwan. It contains a high amount of flavonoids, which have been reported for several health benefits. However, the abundant production usually causes sluggish market sales problems during the harvest period. Hence, it is necessary to develop diversified products of lemon. Black lemon (aged lemon) is a dried citrus fruit, normally made from lemon, which is processed through lightly thermal until it takes on the appearance of blackish-brown leather and becomes dry and preserved. Generally, inflammation is the body`s immune system's response to an irritant and could be classified into chronic and acute two major types of inflammation. Intestinal inflammation is highly correlated with the increased synthesis of nitric oxide (NO) as the consequence of up-regulated inducible nitric oxide synthase (iNOS). The study aimed to determine the total phenolic content and total flavonoid content in aqueous black lemon extract (ABLE) using Folin-Ciocalteu`s method and aluminum chloride colorimetric assay respectively, and the antioxidant capacity of ABLE was determined by FRAP (Ferric reducing antioxidant power). The effects of ABLE on
    lipopolysaccharide (LPS) induced NO production in mouse macrophages
    (RAW264.7) were investigated by measuring the amount of nitrite released into
    the culture medium using the Griess reaction. For the anti-inflammatory effects,
    pro-inflammatory mediators, including iNOS, tumor necrosis factor-α (TNFα), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), were examined by Western blotting. In summary, the result shows that ABLE has a high amount of total phenolic and flavonoid content, besides, it also shows great ferric-reducing ability, therefore, Besides, the results of the analysis of pro-inflammatory factor proteins also showed a great inhibitory effect, especially on the iNOS protein. Overall, based on the results we consider thatABLE might have outstanding antioxidant and anti-inflammation properties, in the future, it has the potential to be an ideal raw material for the development of functional food.

    摘要 I
    Abstract III
    Acknowledgments V
    Table of Contents VI
    List of Tables X
    List of Figures XI
    1. Introduction 1
    1.1 Research background 1
    1.2 Research objective 2
    2. Literature review 3
    2.1 Lemon 3
    2.2 Polyphenols in lemon 5
    2.2.1 Phenolic acids 7
    2.2.2 Flavonoids 7
    2.3 Black lemon 10
    2.4 Maillard reaction 11
    2.4.1 Mechanism of Maillard reaction 11
    2.4.2 Maillard reaction in citrus fruits 11
    2.5 Reactive oxygen species (ROS) 13
    2.5.1 The effects of ROS 13
    2.6 Inflammation 16
    2.6.1 Classification of inflammation 16
    2.6.2 Mechanism of inflammatory response 17
    2.7 Immune system 19
    2.7.1 Macrophages in the immune system 20
    2.7.2 Polarization of macrophages 20
    2.7.2.1 Classical activation 22
    2.7.2.2 Alternative activation 22
    2.7.3 Pattern recognition receptors (PRRs) 23
    2.7.4 Toll-like receptors (TLRs) 25
    2.8 Lipopolysaccharide (LPS) 25
    2.8.1 The structure of LPS 26
    2.8.2 The recognition of LPS 26
    2.9 Cytokines 28
    2.9.1 Tumor necrosis factor-α (TNF-α) 28
    2.9.2 Interleukin-1β (IL-1β) 29
    2.9.3 Interleukin-6 (IL-6) 30
    2.9.4 Interleukin-10 (IL-10) 31
    2.10 NF-κB pathway 32
    2.10.1 Canonical (classical) pathway 32
    2.10.2 Alternative pathway 32
    2.11 Nitric oxide (NO) 34
    2.11.1 Inducible nitric oxide synthase (iNOS) 34
    2.11.2 Cyclooxygenase (COX) and Prostaglandin E2 (PGE2) 35
    2.12 Cell cycle 38
    2.12.1 G0/G1 phase 38
    2.12.2 Synthesis (S) phase 38
    2.12.3 G2 phase 38
    2.12.4 Mitotic (M) phase 39
    2.12.5 Checkpoints 39
    3. Materials and Methods 41
    3.1 Experimental design 41
    3.2 Materials 43
    3.2.2 Reagents 43
    3.2.2.1 Antioxidant study 43
    3.2.2.2 Cell Culture study 43
    3.2.3 Equipment 44
    3.2.4 Antibodies and Kits 44
    3.3 Methods 45
    3.3.1 Black lemon preparation 45
    3.3.2 Preparation of black lemon aqueous extract 45
    3.3.3 Total phenolic content (TPC) 45
    3.3.4 Total flavonoid content (TFC) 46
    3.3.5 Ferric reducing antioxidant power (FRAP) assay 47
    3.3.6 Total polysaccharides content (PC) 48
    3.3.7 Cell Culture 48
    3.3.8 Cell subculture method 49
    3.3.9 Cell freezing (Cell banking) 49
    3.3.10 Cell thawing 50
    3.3.11 Cell counting 50
    3.3.12 MTT assay 50
    3.3.13 Griess assay 51
    3.3.14 ELISA kit assay 51
    3.3.15 Protein extraction 52
    3.3.16 Protein quantification 53
    3.3.17 Western blot analysis 53
    3.3.18 Generation of ROS 55
    3.3.19 Cell cycle 55
    3.3.20 Statistical analysis 56
    4. Results and Discussion 57
    4.1 The extraction solvent and yield 57
    4.2 Antioxidant properties in ABLE 60
    4.3 The amount of polysaccharides content in black lemon 65
    4.4 The effect of different LPS concentrations in RAW264.7 cells 67
    4.5 Cell cytotoxicity test of ABLE 69
    4.6 The effect of ABLE on the morphology of RAW264.7 cells 73
    4.7 Effect of ABLE on nitric oxide production 75
    4.8 The effect of ABLE pre-treatment on the secretion of cytokines 77
    4.8.1 Effect of ABLE on the secretion of TNF-α 77
    4.8.2 Effect of ABLE on the secretion of IL-6 78
    4.8.3 Effect of ABLE on the secretion of IL-10 80
    4.9 The effect of ABLE pre-treatment on the protein expression 82
    4.9.1 Effect of ABLE on the expression of iNOS 82
    4.9.2 Effect of ABLE on the expression of COX-2 84
    4.9.3 Effect of ABLE on the expression of NF-κB 86
    4.9.4 Effect of ABLE on the expression of TNF-α 88
    4.9.5 Effect of ABLE on the expression of IL-6 90
    4.10 Effect of ABLE on the generation of ROS 92
    4.11 Effect of ABLE on cell cycle 94
    5. Conclusion 96
    6. Graph of content 97
    7. References 98

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