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Effect of vegetables on biological function of reactive nitrogen species
iNOS enzyme activity
iNOS protein expression
iNOS enzyme activity
iNOS protein expression
|摘要:||本研究以可能具有降血壓或消炎作用之蔬菜為材料，藉由巨噬細胞模式系統來評估蔬菜水萃取物對活性氮化物生物效應之影響，並以單細胞電泳法探討其對SNP (sodium nitroprusside)及ONOO- (peroxynitrite) 引起RAW 264.7巨噬細胞DNA傷害的影響，以及其對ONOO-誘導LDL氧化修飾的影響，且分析其抗氧化物含量。另外，亦進一步評估乾燥金針花中二氧化硫對一氧化氮生成及細胞基因傷害的影響。
評估蔬菜水萃取物 (200 µg/ml) 的抗氧化力，大致上以蓮藕及茄子較好。在巨噬細胞模式系統中，以豆苗、茄子、絲瓜及新鮮金針花抑制LPS (lipopolysaccharide)誘導巨噬細胞生成NO的能力較好( > 80 %)；而乾燥金針花則較差(-9.2 %)。進一步探討其對iNOS的作用機制，則發現豆苗、茄子會影響iNOS enzyme activity而抑制NO的生成，而絲瓜、新鮮金針花則會影響iNOS enzyme activity與iNOS protein expression而抑制NO的生成。此外，豆苗、茄子、絲瓜及新鮮金針花有抑制SNP誘發RAW 264.7巨噬細胞DNA損傷的能力（>40 %），而乾燥金針花卻有稍促的現象(-10.3 %)；然其亦有抑制ONOO-所誘發的DNA傷害(28 ~ 40 %)，而乾燥金針花則較差 (約0.4 %)。
豆苗、茄子、絲瓜、新鮮金針花及乾燥金針花抑制ONOO-誘導LDL形成TBARS的能力，以2 h的反應時間較好；而在抑制ONOO-誘導LDL形成共軛雙烯的能力，則以0.5 h的反應時間較好。另外，在反應2 h的LDL電泳片上，則發現其抑制LDL氧化修飾的能力效果不佳。於測定蔬菜水萃取物中抗氧化物的方面，豆苗以總多酚化合物(48.20 mg/g)及抗壞血酸(2.20 mg/g)的含量較高，茄子以抗壞血酸(2.25 mg/g)及總花青素(1.67 mg/g)的含量較高，新鮮金針花則以類黃酮(22.44 mg/g)的含量較高。絲瓜及乾燥金針花則在總多酚化合物與總花青素的含量稍不同，具有顯著性差異(P < 0.05)。
探討不同來源之新鮮金針花與乾燥金針花抑制一氧化氮生成的能力，以及評估SO2的存在是否會影響乾燥金針花抑制一氧化氮的生成及細胞基因傷害方面的表現。結果顯示，乾燥金針花與乾燥新鮮金針花清除SNP生成NO的能力較佳(> 30 %)，而新鮮金針花則較差(<10 %)。另外，新鮮金針花與乾燥新鮮金針花抑制LPS誘導巨噬細胞生成NO的能力較佳(> 50 %)，而乾燥金針花則較差(< 6 %)。進一步測定其所含的SO2含量，並將測出的SO2換算成相對濃度之NaHSO3，來探討NaHSO3抑制一氧化氮生成及細胞基因傷害的能力。結果顯示，隨著亞硫酸氫鈉濃度的增加，其清除SNP生成NO的能力隨之增加，且其亦有抑制LPS誘導細胞生成NO的能力，然抑制能力不高(<25 %)。此外，亞硫酸氫鈉於0.8及1.0 µg/ml的濃度之下，會引起細胞基因傷害，而其抑制SNP誘發RAW 264.7巨噬細胞基因損傷的能力則較差，且無顯著性差異(P > 0.05)。因此， SO2於巨噬細胞模式中，對乾燥金針花抑制一氧化氮生成及細胞基因傷害的影響並不顯著。
The objectives of this study were to investigate the effect of vegetables on the biological functions of reactive nitrogen species using macrophage model system, to study the inhibitory effect of vegetables on sodium nitroprusside (SNP) or peroxynitrite (ONOO-) induced DNA damage in RAW 264.7 macrophage using single cell electrophoresis, to study the inhibitory effect of vegetables on ONOO- mediated in LDL oxidation, and to analysis their antioxidants content. In addition, the effect of sulfur dioxide in dried daylily on the generation of nitric oxide and DNA damage in cells was also studied. The water extracts (200 µg/ml) of indian lotus and eggplant had good antioxidant activity. In macrophage model system, bean sprout, eggplant, vegetable sponge and fresh daylily exhibited the inhibitory effect on nitric oxide generation induced by lipopolysaccharide (LPS) (>80 %) whereas the dried daylily had enhancing effect (-9.2 %). Further study was conducted on some selected vegetable extracts to elucidate their reaction mechanisms on iNOS using macrophage model system. The results showed that bean sprout and eggplant decreased the production of NO by the inhibition of iNOS enzyme activity; however, vegetable sponge and fresh daylily decreased the production of NO by the inhibition of both iNOS enzyme activity and iNOS protein expression. In addition, bean sprout, eggplant, vegetable sponge and fresh daylily showed the inhibitory effect on DNA damage in RAW 264.7 macrophage induced by SNP (>40%) but the dried daylily increased the DNA damage (-10.3 %). However, bean sprout, eggplant, vegetable sponge, and fresh daylily also showed the inhibitory effect on DNA damage in RAW 264.7 macrophage induced by ONOO- (28~40 %) but the dried daylily did not (about 0.4 %). The better inhibitory effect of bean sprout, eggplant, vegetable sponge, fresh daylily and dried daylily on the production of TBARS and conjugated diene in ONOO- mediated LDL oxidation was by incubation for 2 h and 0.5 h, respectively. In addition, the result of electrophoretic pattern showed that bean sprout, eggplant, vegetable sponge, fresh daylily and dried daylily had no inhibitory effect on LDL oxidation when LDL was incubated with ONOO- for 2 h. The analysis of antioxidant in water extracts from vegetables showed that bean sprout had higher content of total polyphenol compounds (48.20 mg/g) and ascorbic acid (2.20 mg/g), eggplant had higher content of ascorbic acid (2.25 mg/g) and total anthocyanins (1.67 mg/g), and fresh daylily had higher content of flavonoids (22.44 mg/g). Vegetable sponge and dried daylily had significantly different (P<0.05) content in total polyphenol compounds and total anthocyanins. The results showed that dried daylily and dried fresh daylily had stronger scavenging effect on the generation of nitric oxide by SNP (>30 %) but the fresh daylily did not. In macrophage model system, fresh daylily and dried fresh daylily exhibited the inhibitory effect on the generation of nitric oxide induced by LPS (>50 %) but the dried daylily did not (<6 %). Sodium hydrogen sulfite with equal amount of sulfur dioxide in dried daylily was used to study its inhibitory effect on the generation of nitric oxide and DNA damage in cells. The results showed that the scavenging effect of sodium hydrogen sulfite on the generation of nitric oxide by SNP was in a dose-dependent manner, and it also exhibited a weak inhibitory effect on the generation of nitric oxide induced by LPS (<25 %) in RAW 264.7 macrophages. Sodium hydrogen sulfite had no effect on SNP induced DNA damage in RAW 264.7 macrophages; however, it induced DNA damage at the concentration of 0.8 and 1.0 µg/ml. Thus, sulfur dioxide in dried daylily had no significantly effect on the generation of nitric oxide and DNA damage in cells. Based on the results of this study, the water extracts of bean sprout, eggplant, vegetable sponge and fresh daylily had good inhibitory effect on the biological functions of reactive nitrogen species, and it should be beneficial for human health to intake those vegetables. In addition, sulfite is a bleaching agent for processing of dried daylily. Although sulfur dioxide is volatilized during heating, it may also residue in food and influence the balance of antioxidant system. Thus, the intake of dried daylily should be in a proper quantity.
|Appears in Collections:||食品暨應用生物科技學系|
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