Comparison of Antioxidant Activities of Three Kinds of Juices and Their Protective Effects on Oxidative Damage of Colon Cell NCM460
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摘要: 比较刺梨汁（Rosa roxburghii Tratt juice，RRTJ）、石榴汁（Pomegranate juice，PJ）以及蓝莓汁（Blueberry juice，BJ）的活性成分含量以及抗氧化活性，探究三种果汁对葡聚糖硫酸钠盐（Dextran sulfate sodium，DSS）诱导人正常结肠上皮细胞NCM460氧化损伤的保护作用。结果表明，三种果汁中共同含有的生物活性成分有28种，其中刺梨汁的总多酚、总黄酮含量显著高于石榴汁和蓝莓汁（P<0.05），分别为22.77和12.04 mg/mL；同时，刺梨汁对ABTS+·、DPPH·的清除能力显著高于石榴汁和蓝莓汁（P<0.05），半数清除率（Half scavenging rate，IC50）分别为4.00±0.32和10.03±0.51 μL/mL；Pearson相关性分析表明果汁的总多酚含量与ABTS+·清除能力呈正相关（P<0.05）。此外，刺梨汁缓解DSS诱导NCM460细胞氧化损伤的能力最强，2 μL/mL刺梨汁即能使氧化损伤的NCM460细胞活力恢复到与对照组相当的水平，降低DSS引起的细胞中活性氧（Reactive oxygen species，ROS）水平。实验表明果汁中的总多酚含量与抗氧化能力呈正相关；刺梨汁的总多酚和总黄酮含量高于蓝莓汁和石榴汁，抗氧化活性和缓解DSS诱导NCM460细胞氧化损伤的能力最强，具有深入研究开发的潜力。Abstract: To compare the contents of active ingredients and antioxidant activity of Rosa roxburghii Tratt juice (RRTJ), pomegranate juice (PJ) and blueberry juice (BJ) and investigate the protective effects of three kinds of fruit juices against dextran sulfate sodium (DSS)-induced oxidative damage in human normal colonic epithelial cells NCM460. There were 28 bioactive components in the three juices. The total polyphenols and total flavonoids of RRTJ were significantly higher than those in the PJ and BJ (P<0.05), 22.77 and 12.04 mg/mL, respectively. Meanwhile, the ABTS+· and DPPH· scavenging abilities of the juice were significantly higher than those in the PJ and BJ (P<0.05), and the half scavenging rate (IC50) was 4.00±0.32 and 10.03±0.51 μL/mL, respectively. Pearson correlation analysis indicated that the total polyphenol content of the juice was positively correlated with the ABTS+· scavenging ability (P<0.05). In addition, RRTJ had the strongest ability to alleviate DSS-induced oxidative damage in NCM460 cells, and 2 μL/mL RRTJ could restore the viability of oxidatively damaged NCM460 cells to a level comparable to the control and reduce the level of reactive oxygen species (ROS) in DSS-induced cells. The results showed that the content of total polyphenols in fruit juice was positively correlated with antioxidant capacity. The content of total polyphenols in juice was positively correlated with antioxidant capacity, and the antioxidant activity and the ability to alleviate DSS-induced oxidative damage in NCM460 cells were the strongest in vitro, which had the potential for in-depth research and development.
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图 1 三种果汁的总多酚和总黄酮含量
注：不同果汁（RRTJ：刺梨汁；PJ：石榴汁；BJ：蓝莓汁）同一指标标不同小写字母表示具有显著性差异（P<0.05）；表3同。
Figure 1. Total polyphenol and total flavonoid contents of three kinds of fruit juices
图 2 三种果汁对ABTS+·、DPPH·、·OH的清除率
Figure 2. Scavenging rate of ABTS+·, DPPH·, ·OH of three kinds of fruit juices
图 3 DSS对NCM460细胞存活率的影响
注：*表示与对照相比存在显著性差异，P<0.05；**表示与对照相比存在极显著性差异，P<0.01；图4同。
Figure 3. Effect of DSS on the survival rate of NCM460 cells
图 4 三种果汁对NCM460细胞存活率的影响
Figure 4. Effects of three kinds of juices on the survival rate of NCM460 cells
图 5 三种果汁对NCM460细胞氧化损伤的保护作用
注：#表示与对照组相比具有显著性差异（P<0.05），##表示与对照组相比具有极显著性差异（P<0.01）；*表示与DSS组相比具有显著性差异（P<0.05），**表示与DSS组相比具有极显著性差异（P<0.01）；图6同。
Figure 5. Protective effects of three kinds of fruit juices on oxidative damage in NCM460 cells
图 6 三种果汁对NCM460细胞内ROS水平的影响
Figure 6. Effects of three fruit juices on ROS levels in NCM460 cells
表 1 梯度洗脱程序
Table 1. Gradient elution procedure
序号 时间 A相（%） B相（%） 1 0 95 5 2 2 70 30 3 5 30 70 4 7 10 90 5 8 10 90 6 8.1 95 5 7 10 95 5 
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表 2 三种果汁中的活性成分
Table 2. Active ingredients in three kinds of fruit juices
序号 分类 化合物 分子式 分子量 保留时间（min） 质荷比（m/z） 模式 峰面积比:RRTJ/RRTJ 峰面积比:PJ/RRTJ 峰面积比:BJ/RRTJ 1 酚酸 儿茶素 C15H14O6 290.07896 4.557 289.0718 [M-H]− 1 0.0185381 0.0047719 2 酚酸 对香豆酸 C9H8O3 164.04727 4.627 163.0401 [M-H]− 1 0.3546840 0.0243441 3 酚酸 异香草酸 C8H8O4 168.04224 3.102 170.0964 [M+H]+ 1 21.1876641 18.9522201 4 酚酸 咖啡酸 C9H8O4 180.04226 3.793 179.0349 [M-H]− 1 0.9439670 9.4652850 5 酚酸 阿魏酸 C10H10O4 194.05783 5.681 193.0870 [M-H]− 1 1.6777904 47.0191357 6 酚酸 芥子酸 C11H12O5 224.06844 4.527 225.1495 [M+H]+ 1 11.227866 142.189311 7 酚酸 鞣花酸 C14H6O8 302.00618 5.593 303.0133 [M+H]+ 1 0.1997369 0.0140473 8 酚酸 对香豆酰奎尼酸 C16H18O8 338.10026 4.889 337.0930 [M-H]− 1 0.0588202 1.5330073 9 酚酸 新绿原酸 C16H18O9 354.09494 3.74 353.0879 [M-H]− 1 0.0036370 0.5928340 10 酚酸 表儿茶素 C15H14O6 290.07893 4.953 289.0718 [M-H]− 1 0.0641652 0.0731626 11 黄酮类 紫云英苷 C21H20O11 448.1004 4.401 447.0569 [M-H]− 1 0.0021714 0.0020716 12 黄酮类 芦丁 C27H30O16 610.1536 5.657 609.1976 [M-H]− 1 0.3958080 56.4030733 13 黄酮类 染料木苷 C21H20O10 432.10564 4.756 433.1677 [M+H]+ 1 372.1262714 3.4947074 14 生物碱 葫芦巴碱 C7 H7NO2 105.02136 0.827 136.8267 [M-H]− 1 35.9551281 1.2682148 15 有机酸 富马酸 C4H4 O4 116.01081 0.879 115.0039 [M-H]− 1 0.2096278 0.0881666 16 有机酸 衣康酸 C5 H6 O4 130.0266 0.948 129.0192 [M-H]− 1 1.03847909 1.2074671 17 有机酸 DL-苹果酸 C4H6O5 134.02149 1.061 133.0143 [M-H]− 1 0.7183395 0.1855467 18 有机酸 反乌头酸 C6 H6 O6 174.01642 0.912 173.0095 [M-H]− 1 1.5748353 1.8560623 19 有机酸 维生素C C6 H8 O6 174.01636 1.49 175.0247 [M-H]− 1 0.4208721 0.6294469 20 有机酸 柠檬酸 C6 H8 O7 192.0633 1.278 191.0197 [M-H]− 1 0.1741608 0.6592146 21 有机酸 奎宁酸 C7 H12 O6 192.06341 0.832 191.0560 [M-H]− 1 0.0798837 8.4139563 22 有机酸 脱落酸 C15 H20 O4 264.13628 7.092 265.1047 [M+H]+ 1 0.3879990 5.3032785 23 糖苷 长寿花糖甙 C19 H30 O8 386.19405 5.108 385.1862 [M-H]− 1 0.1444265 0.7048008 24 氨基酸 赖氨酸 C6 H14N2O2 146.10549 0.695 145.0983 [M-H]− 1 17.3911049 4.8403282 25 氨基酸 組氨酸 C6 H9 N3 O2 155.06944 0.703 154.4484 [M-H]− 1 15.7036029 4.4824619 26 氨基酸 L-酪氨酸 C9 H11 N O3 181.07389 1.294 163.0400 [M-H]− 1 8.3561917 1.5198959 27 氨基酸 DL-精氨酸 C6H14N4O2 174.11154 0.715 173.0093 [M-H]− 1 5.3336296 52.6650510 28 氨基酸 异亮氨酸 C6H13NO2 131.09461 1.379 130.0876 [M-H]− 1 1.5481378 0.8577223
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表 3 三种果汁抗氧化活性的IC50
Table 3. IC50 of antioxidant activity of three kinds of fruit juices
IC50（μL/mL） RRTJ PJ BJ ABTS+· 4.00±0.32b 40.74±0.72a 39.37±0.74a DPPH· 10.03±0.51c 362.77±71.48b 840.43±73.57a ·OH 5.92±0.46b 19.79±0.85a 5.67±0.82b
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表 4 活性成分含量与抗氧化之间的相关性
Table 4. Correlation between active ingredient content and antioxidation
r 总多酚 总黄酮 IC50 of ABTS+· IC50 of DPPH· IC50 of ·OH 总多酚 1.000 总黄酮 0.994 1.000 IC50 of ABTS+· −0.999* −0.988 1.000 IC50 of DPPH· −0.769 −0.697 0.800 1.000 IC50 of ·OH −0.558 −0.642 0.512 −0.102 1.000 注：*表示存在显著性，P<0.05。
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