Ultrasonic-Assisted Extraction Optimization of Antioxidant Products from Hyrtios erectus and Its Antioxidant Activity
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摘要: 为探索海绵动物抗氧化提取物的提取工艺及提取物的抗氧化活性，以H. erectus海绵乙醇提取物的DPPH自由基清除率为响应值，分别考察超声温度、超声时间和超声功率3个影响因素，通过Box-Behnken响应面设计确定最佳超声提取工艺。以该工艺提取物为实验材料，分析其对DPPH自由基、ABTS+?和?OH的清除效果，通过构建H2O2氧化损伤模型研究提取物对氧化损伤L02细胞的活力和对H2O2氧化应激胞内ROS含量的影响。结果表明：可操作的最佳工艺为超声温度57 ℃，超声时间60 min，超声功率490 W，在此条件下，提取物DPPH自由基清除率为（61.98±1.52）%，与预测值62.16%吻合度较好，该提取物对DPPH自由基、ABTS+?和?OH具有良好的清除效果，提取物处理组细胞活力均显著高于模型组（P<0.05），且细胞内ROS荧光强度均极显著低于模型组（P<0.01）。总之，该工艺提取物具有较广泛的抗氧化活性，对H2O2氧化损伤的L02细胞具有保护作用，该研究可为抗氧化食品添加剂的研发提供理论支撑。Abstract: To explore the process of extraction and antioxidant activity of products from marine sponge, three influencing factors, ultrasonic temperature, ultrasonic time, and ultrasonic power were investigated respectively taking DPPH radicals scavenging rate of ethanol extracts from H. erectus as the response value, and the optimal ultrasonic-assisted extraction process was determined by Box-Behnken design. The extract obtained from H. erectus by the best ultrasonic-assisted process was detected for antioxidant activity, which included the scavenging effect on DPPH radicals, ABTS+? and ?OH. The effects of the extract on viability of oxidative damage L02 cells and content of intracellular ROS were detected by constructing a cell model of H2O2 induced oxidative damage. The results showed that the optimized process conditions were as follows: ultrasonic temperature was 57 ℃, ultrasonic time was 60 min, and ultrasonic power was 490 W. Under these conditions, the DPPH scavenging rate of the extract was (61.98±1.52)%, which agreed well with the predicted value of 62.16%. The extract showed good scavenging effects on DPPH radical, ABTS+? and ?OH. The cell viability of treated groups was significantly higher than that of the model group (P<0.05), and the intensity of intracellular ROS fluorescence was significantly lower than that of the model group (P<0.01). In general, the product from H. erectus had a wide range of antioxidant activity, and it had a protective effect on H2O2 induced oxidative damage in L02 cells. This study provides theoretical support for the research and development of antioxidant food additives.
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Key words:
- response surface /
- marine sponge /
- anti-oxidation /
- free radical /
- reactive oxygen species (ROS)
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图 1 超声温度对DPPH自由基清除率和粗提物得率的影响
Figure 1. Effect of ultrasonic temperature on the scavenging rate of DPPH free radical and yield of crude extract
图 2 超声时间对DPPH自由基清除率和粗提物得率的影响
Figure 2. Effect of ultrasonic time on the scavenging rate of DPPH free radical and yield of crude extract
图 3 超声功率对DPPH自由基清除率和粗提物得率的影响
Figure 3. Effect of ultrasonic power on the scavenging rate of DPPH free radical and yield of crude extract
图 4 各因素交互作用对DPPH自由基清除率的响应面和等高线图
Figure 4. Response surface and contour figures of the interaction effects of various factors on the scavenging rate of DPPH free radicals
图 5 提取物对3种自由基的清除效果
Figure 5. The scavenging effect of the extract on three free radicals
图 6 提取物对H2O2氧化损伤L02细胞活力的影响
Figure 6. Effects of extracts on the viability of L02 cells damaged by H2O2 oxidation
注：*表示差异显著（P<0.05），**表示差异极显著（P<0.01）；+表示添加，−表示未添加；图7同。
图 7 提取物对H2O2氧化应激L02细胞内ROS的影响
Figure 7. Effect of extracts on reactive oxygen species in L02 cells with H2O2-induced oxidative damage
表 1 Box-Behnken设计的因素与水平
Table 1. Factors and levels of Box-Behnken design
因素 编码 水平 ?1 0 1 超声温度（℃） A 40 50 60 超声时间（min） B 50 60 70 超声功率（W） C 400 500 600 
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表 2 Box-Behnken试验设计结果
Table 2. Results of Box-Behnken experiments design
试验号 A超声温度 B超声时间 C超声功率 Y DPPH自由基清除率
（%）1 0 1 ?1 48.08±2.25 2 0 1 1 41.69±2.59 3 ?1 ?1 0 38.82±2.41 4 1 ?1 0 46.31±1.70 5 0 0 0 62.03±1.11 6 1 1 0 41.65±1.25 7 ?1 1 0 42.76±2.21 8 0 0 0 61.80±1.26 9 1 0 ?1 45.36±1.59 10 0 ?1 1 44.22±2.50 11 ?1 0 ?1 46.01±3.34 12 1 0 1 44.31±1.01 13 0 0 0 61.79±1.70 14 0 0 0 61.98±2.82 15 ?1 0 1 37.47±1.33 16 0 ?1 ?1 47.49±2.13 17 0 0 0 62.09±3.15 
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表 3 回归模型方程的方差分析
Table 3. Variance analysis of regression model equation
方差来源 平方和 自由度 均方 F值 P 显著性 回归模型 1297.09 9 144.12 3800.45 <0.0001 ** A-超声温度 19.75 1 19.75 520.82 0.0001 ** B-超声时间 0.88 1 0.88 23.32 0.019 * C-超声功率 46.32 1 46.32 1221.46 <0.0001 ** AB 18.49 1 18.49 487.58 <0.0001 ** AC 14.03 1 14.03 369.84 <0.0001 ** BC 2.43 1 2.43 64.17 <0.0001 ** A2 492.73 1 492.73 12993.24 <0.0001 ** B2 321.28 1 321.28 8472.16 <0.0001 ** C2 258.32 1 258.32 6811.96 <0.0001 ** 残差 0.27 7 0.038 ? ? ? 失拟项 0.19 3 0.064 3.43 0.1323 N 净误差 0.074 4 0.019 ? ? ? 总离差 1297.36 16 ? ? ? ? R2=0.9998，R2adj=0.9995 注：*代表P<0.05，**代表P<0.01，N代表差异无统计学意义，“?”表示无此项。 
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