臺灣博碩士論文加值系統

薑黃素是一種從薑黃中提取的一種黃色天然色素，具有多種藥理作用，包括消炎，抗氧化，抗細胞增生和抗血管新生等活性。而薑黃酮則是薑黃油的主要成分，具有抗發炎之效果。先前的研究表明薑黃素在骨細胞中具有良好的生物活性，但是沒有找到薑黃酮對骨細胞作用之相關研究。基於這些文獻綜述，本研究的目的是研究薑黃萃取物（乙醇萃取和超臨界萃取）對骨細胞的作用，並且希望發現薑黃酮是否具有與薑黃素相似的生物作用。
本研究的薑黃粉末為行政院農業委員會農業試驗所嘉義農業試驗分所提供的台灣品種薑黃，再用兩種萃取方式獲得薑黃萃取液，一種是利用超臨界流體萃取（SFE）方式萃取薑黃粉，並通過GC-MS確定其主要成分為“薑黃酮”。此外，亦用乙醇作為溶劑萃取薑黃粉（EE），並通過HPLC發現其主要成分為“薑黃素”。之後，觀察比較這兩種萃取物（EE和SFE）在RAW264.7小鼠巨噬細胞和7F2小鼠成骨細胞的細胞毒殺性，並選擇細胞存活率超過80%的萃取物濃度作細胞實驗。此外，利用DPPH和ABTS測定兩種薑黃萃取液的抗氧化活性；並且使用H2O2誘導7F2小鼠成骨細胞損傷並測定細胞內ROS的生成量與細胞老化比例。再利用LPS刺激RAW264.7小鼠巨噬細胞發炎並測定薑黃萃取液之抗發炎活性，並使用脂質分化培養液(ADM)誘導7F2小鼠成骨細胞生成脂質來研究抑制脂肪分化之能力。最後，通過即時定量PCR檢測COX-2, FAS和FABP4的基因表現。
總體而言，實驗結果表明，薑黃萃取物（EE ＆ SFE）具有良好的抗氧化活性，並且隨著萃取物濃度增長而逐漸抑制LPS誘導的RAW264.7小鼠巨噬細胞中的COX-2基因表現和NO釋放。此外，薑黃提取物（EE ＆ SFE）可以通過減少細胞內ROS的生成來抑制細胞衰老，並通過下調FAS和FABP4基因表達來減少7F2小鼠成骨細胞中的脂質堆積。最後，兩種薑黃萃取物相比之下，薑黃萃取物EE的生物活性高於SFE。因此，乙醇萃取可能是從薑黃中分離生物活性化合物的適合方法。

Curcumin, (1,7-Bis(4-hydroxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione), a yellow pigment extracted from Curcuma longa, possesses diverse pharmacologic effects including anti-inflammatory, antioxidant, anti-proliferative and antiangiogenic activities. Turmerone, (2-Methyl-6-(4-methylphenyl)-2-hepten-4-one), is one of lipophilic components in turmeric extract and that has anti-inflammatory effects.
Previously, our laboratory has extracted the turmeric powder by supercritical fluid extraction (SFE), and identified its main composition “turmerone” by GC-MS. Besides, I used ethanol as solvent for the extraction of turmeric powder (EE), and found its main composition, curcumin, by HPLC. Hence, I used these two extracts (EE and SFE) to compare their cytotoxicity in RAW264.7 mouse macrophages and 7F2 osteoblasts and the concentrations of turmeric extracts with cell viability over 80% were selected for DPPH and ABTS assays. Moreover, intracellular ROS production and cell-senescence were investigated using H2O2-induced 7F2 osteoblasts. LPS-stimulated RAW264.7 mouse macrophages were used to measure anti-inflammatory activity, and adipogenic differentiation medium (ADM)-treated 7F2 osteoblasts were used to investigate anti-adipogenic ability. Finally, we measured the gene expressions of COX-2, FAS and FABP4 by quantitative real-time PCR.
Previous studies have demonstrated that curcumin has good biological activities in bone cells, but there is no similar study to investigate the effect of turmerone on bone cells. Based on these literature review, the objective of this study is to investigate the effect of the turmeric extracts (EE and SFE) on bone cells, and we are keen to find whether turmerone has similar biological effects as curcumin.
So far, the results indicated that turmeric extracts (EE & SFE) showed good antioxidant activities and could suppress NO release and COX-2 mRNA expression in RAW264.7 mouse macrophages dose dependently. In addition, turmeric extracts (EE & SFE) could suppress intracellular ROS production, cell aging and lipid accumulation in 7F2 mouse osteoblasts. The adipogenesis-related gene expressions, FAS and FABP4, were reduced by the treatment of turmeric extracts. In comparison, the bioactivities of turmeric extract (EE) was higher than SFE. Therefore, ethanol extraction maybe the better way to isolate bioactive compounds from Curcuma longa.

中文摘要………………………………………………………………………………1
Abstract……………………………………………………………………………….3
致謝……………………………………………………………………………………5
Index…………………………………………………………………………………..6
Figure index…………………………………………………………………………..9
Table index…………………………………………………………………………..11
Scheme index………………………………………………………………………...12
Chapter 1 Introduction
1.1 Bone structure………………………………………………………………...13
1.2 Oxidative stress and anti-oxidant…………………………………………... 14
1.3 Inflammation…………………………………………………........................16
1.4 Adipogenic differentiation…………………………………………………...18
1.5 Curcumin and turmerone……………………………………………………21
1.6 Aim of thesis…………………………………………………………………..24
1.7 Experimental design………………………………………………………….25
Chapter 2 Materials and Methods
2.1 Materials………………………………………………………………………27
2.2 Cell culture……………………………………………………………………27
2.3 Extract of turmeric powder………………………………………………….28
2.4 Total polyphenolic assay……………………………………………………...28
2.5 Determination of triterpenoid………………………………………………..28
2.6 Antioxidant activity by DPPH assay…………………………………………29
2.7 Radical scavenging activity by ABTS assay…………………………………30
2.8 Cell viability assay…………………………………………………………….30
7
2.9 Nitrite assay…………………………………………………………………...31
2.10 Detection of oxygen species (ROS) level by DCF-DA staining……………31
2.11 DAPI Staining………………………………………………………………..32
2.12 Senescence-Associated β-Galactosidase (SA-β-Gal) Staining…………….33
2.13 Oil Red O Staining…………………………………………………………..34
2.14 Triglyceride assay……………………………………………………………34
2.15 Quantitative real-time PCR………………………………………………...35
2.16 HPLC………………………………………………………………………...35
2.17 Gas chromatography–mass spectrometry (GC-MS)………………………36
2.18 Statistical analysis…………………………………………………………...37
Chapter 3 Results
3.1 HPLC analysis of turmeric extracts (ETOH/SFE)…………………………38
3.2 Total polyphenol content in turmeric extracts………………………………40
3.3 Total triterpenoid content in turmeric extracts……………………………..41
3.4 Antioxidant activity by DPPH assay…………………………………………42
3.5 Antioxidant activity by ABTS assay…………………………………………43
3.6 The effect of the turmeric extracts (EE & SFE) on cell viabilities of
RAW264.7 mouse macrophages and 7F2 mouse osteoblasts…………………..44
3.7 The effect of EE and SFE turmeric extracts on nitrite production of
RAW264.7 mouse macrophages…………………………………………………47
3.8 The effect of turmeric extracts (EE&SFE) on gene expression of LPS
induced inflammatory factors…………………………………………………..48
3.9 The effect of turmeric extracts (EE & SFE) on ROS production of 7F2 mouse
osteoblasts………………………………………………………………………...50
3.10 The effect of turmeric extracts (EE & SFE) on cell senescence of 7F2 mouse
osteoblasts………………………………………………………………………...54
8
3.11 The effect of turmeric extracts (EE&SFE) on lipid accumulation of ADMtreated 7F2 mouse osteoblasts……………………………………………………56
3.12 The effect of turmeric extracts (EE & SFE) on triglyceride synthesis in
ADM-induced 7F2 mouse osteoblasts…………………….……………………..60
3.13 The effect of turmeric extracts (EE&SFE) on gene expression of adipogenic
markers in 7F2 mouse osteoblasts……………………………………………….62
Chapter 4 Discussion………………………………………………………………..64
Chapter 5 Conclusion……………………………………………………………….69
Reference…………………………………………………………………………….70

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