猪血中超氧化物歧化酶（SOD）和血红素的提取及SOD的应用研究
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摘要
我国猪血资源非常丰富,猪血的蛋白质含量高、氨基酸组成平衡,是优质的蛋白来源。猪血中以红细胞为主,红细胞中最主要的蛋白是血红蛋白,含量占血细胞蛋白总量的90%以上。开发猪血资源不仅可增加蛋白质饲料来源、增加用于食品、医学等领域的原料资源,又可减少屠宰废弃物对环境的污染,具有很好的经济和社会效益。本论文以猪血为原料提取SOD和血红素,进行初步鉴定、储存过程中的稳定性实验,并对SOD的体外抗氧化活性及其对肿瘤细胞凋亡的影响进行研究。
1 SOD和血红素提取工艺条件的优化
采用超声波破碎细胞膜,SOD比活力和血红素提取率都较高的超声条件为：超声功率70 W、超声时间15 min及超声温度40℃；利用Box-Behnken的中心组合旋转设计试验,Design-Expert软件处理数据,得出SOD的最佳提取条件：热变温度65.2℃、热变时间26.1 min、0.1 mol/L氯化铜添加0.423mL/15mL溶血液；血红素的最佳提取条件为：酸性丙酮添加量为血红蛋白的5.66倍,酸酮比为3%,抽提时间为42.00 min时血红素提取率达到最大值为0.225 g／100 mL。
2 SOD和血红素的初步鉴定
SOD提取样品的紫外光谱分析最大吸收峰为265 nm,与文献报道一致；血红素标品和样品进行紫外光谱、红外图谱和高效液相色谱分析确定提取物为血红素。选择甲醇：水为80：20,在所选流动相和仪器条件下,检出限、精密度和稳定性均良好,血红素的纯度为72%。
3 SOD和血红素的储存稳定性研究
SOD和血红素在储存过程中,冷冻保存有利于SOD和血红素的稳定。储存20d时,冷冻保存SOD的相对活力为90%,冷藏保存相对活力为85%,室温避光保存为82%,室温光照相对活力为80.25%；储存9周后,冷冻保存的血红素相对含量为91.58%,冷藏保存、室温避光保存和室温光照保存的相对含量分别为88.61%、83.46%和81.75%。
4 SOD的体外抗氧化活性及对小鼠荷瘤sp2／0细胞凋亡的影响
SOD的体外抗氧化实验中以Vc作为对照品,检测SOD的体外抗氧化活性,由实验结果可知SOD有一定的抗氧化活性,但其抗氧化活性比同浓度的Vc低。
采用研究细胞凋亡的最基本的方法：凋亡细胞的形态学观察。由本实验可见未加SOD的小鼠荷瘤sp2／0细胞生长良好,表现为圆形；经不同浓度SOD作用一段时间后的小鼠荷瘤sp2／0细胞其细胞核明显固缩、碎裂,表现为凋亡形态,且随着作用浓度的增加,细胞核固缩碎裂的程度增加。SOD在体外可在一定程度上抑制小鼠荷瘤sp2／0细胞的增殖,呈时间-效应和剂量-效应的关系,能够诱导小鼠荷瘤sp2／0细胞的凋亡。
Pig blood is very rich in our country, it is rich in protein and amino acid is balance, it is the source of high-quality protein. Red blood cell is the principal components in pig blood, the main protein in red blood cell is hemoglobin, account for more than 90%. To develop pig blood resources has good economic and social benefits, it is not only can increase the protein of feed sources, increase raw material resources for food, medicine and other fields, but also to reduce the pollution of environment from animal waste. In this thesis, SOD and heme were extracted from pig blood, structural identification, stability in stored procedure of SOD and heme, the in vitro antioxidant activity and the effect on mouse-borne tumor cell apoptosis of SOD were studied.
1 The optimization extraction condition of SOD and heme
When broken the cell membrane by ultrasonic, the optimum ultrasonic conditions for high SOD specific activity and high heme extraction rate were:ultrasonic power 70 W, ultrasonic time 15min and ultrasonic temperature 40℃; Using Box-Behnken of the central composite rotary design test, Design-Expert software to process data, the best extraction conditions obtained SOD:heating temperature 65.2℃, heating time 26.1 min, copper chloride solution of 0.1 mol/L added 0.423 mL/15 mL dissolve blood; the optimum extraction conditions of heme:acidic acetone addition level was 5.66 times that of hemoglobin, ratio of hydrochloric acid and acetone was 3%, the extraction time was 42.00 min, the maximum extraction rate of heme was 0.225 g/100 mL.
2 Initial structure identification of SOD and heme
Analyzed extracted SOD samples by UV spectrum, the maximum absorption peak is 265 nm, the result was consistent with those reported; compared the spectrogram of standard heme and samples heme by UV spectrum, Infrared spectrum and high performance liquid chromatography, the extraction sample was proved to be heme. Under the choosen mobile phase(methanol:water was 80:20) and equipment condition, detection limits, precision and stability was good, purity of heme was 72%.
3 Storage stability of SOD and heme
Freezing storage was conducive to the stability of SOD and heme in storage. In freezing storage, chill storage, room temperature and dark storage, room temperature in daylight storage, the relatively activity of SOD were 90%,85%,82%and 80.25%after 20 h. In freezing storage, chill storage, room temperature and dark storage, room temperature in daylight storage, the relatively contents of heme were 91.58%,88.61%,83.46% and 81.75% after 9 weeks.
4 Antioxidant activity and effects on mouse-borne tumor sp2/0 cell apoptosis of SOD
The antioxidant activity of SOD was detected in vitro, using Vc as the reference standard, the results showed that SOD had good antioxidant activity, but lower than that of Vc.
Observation of cell morphology is the basic method of apoptosis study. It was indicated that mouse-borne tumor SP2/0 cell grew well without SOD, showing round; mouse-borne tumor SP2/0 cell was significant nuclear condensation, fragmentation, showing the apoptotic morphology with different SOD concentration after a while, and with the increasing of the concentration, the extent of nuclear condensation and fragmentation increased. SOD can inhibit mouse-borne tumor SP2/0 cell proliferation to some extent in vitro, and showed time-effect and dose-effect relationship, SOD could induce mouse-borne tumor SP2/0 cell apoptosis.
1 SOD和血红素提取工艺条件的优化
采用超声波破碎细胞膜,SOD比活力和血红素提取率都较高的超声条件为：超声功率70 W、超声时间15 min及超声温度40℃；利用Box-Behnken的中心组合旋转设计试验,Design-Expert软件处理数据,得出SOD的最佳提取条件：热变温度65.2℃、热变时间26.1 min、0.1 mol/L氯化铜添加0.423mL/15mL溶血液；血红素的最佳提取条件为：酸性丙酮添加量为血红蛋白的5.66倍,酸酮比为3%,抽提时间为42.00 min时血红素提取率达到最大值为0.225 g／100 mL。
2 SOD和血红素的初步鉴定
SOD提取样品的紫外光谱分析最大吸收峰为265 nm,与文献报道一致；血红素标品和样品进行紫外光谱、红外图谱和高效液相色谱分析确定提取物为血红素。选择甲醇：水为80：20,在所选流动相和仪器条件下,检出限、精密度和稳定性均良好,血红素的纯度为72%。
3 SOD和血红素的储存稳定性研究
SOD和血红素在储存过程中,冷冻保存有利于SOD和血红素的稳定。储存20d时,冷冻保存SOD的相对活力为90%,冷藏保存相对活力为85%,室温避光保存为82%,室温光照相对活力为80.25%；储存9周后,冷冻保存的血红素相对含量为91.58%,冷藏保存、室温避光保存和室温光照保存的相对含量分别为88.61%、83.46%和81.75%。
4 SOD的体外抗氧化活性及对小鼠荷瘤sp2／0细胞凋亡的影响
SOD的体外抗氧化实验中以Vc作为对照品,检测SOD的体外抗氧化活性,由实验结果可知SOD有一定的抗氧化活性,但其抗氧化活性比同浓度的Vc低。
采用研究细胞凋亡的最基本的方法：凋亡细胞的形态学观察。由本实验可见未加SOD的小鼠荷瘤sp2／0细胞生长良好,表现为圆形；经不同浓度SOD作用一段时间后的小鼠荷瘤sp2／0细胞其细胞核明显固缩、碎裂,表现为凋亡形态,且随着作用浓度的增加,细胞核固缩碎裂的程度增加。SOD在体外可在一定程度上抑制小鼠荷瘤sp2／0细胞的增殖,呈时间-效应和剂量-效应的关系,能够诱导小鼠荷瘤sp2／0细胞的凋亡。
Pig blood is very rich in our country, it is rich in protein and amino acid is balance, it is the source of high-quality protein. Red blood cell is the principal components in pig blood, the main protein in red blood cell is hemoglobin, account for more than 90%. To develop pig blood resources has good economic and social benefits, it is not only can increase the protein of feed sources, increase raw material resources for food, medicine and other fields, but also to reduce the pollution of environment from animal waste. In this thesis, SOD and heme were extracted from pig blood, structural identification, stability in stored procedure of SOD and heme, the in vitro antioxidant activity and the effect on mouse-borne tumor cell apoptosis of SOD were studied.
1 The optimization extraction condition of SOD and heme
When broken the cell membrane by ultrasonic, the optimum ultrasonic conditions for high SOD specific activity and high heme extraction rate were:ultrasonic power 70 W, ultrasonic time 15min and ultrasonic temperature 40℃; Using Box-Behnken of the central composite rotary design test, Design-Expert software to process data, the best extraction conditions obtained SOD:heating temperature 65.2℃, heating time 26.1 min, copper chloride solution of 0.1 mol/L added 0.423 mL/15 mL dissolve blood; the optimum extraction conditions of heme:acidic acetone addition level was 5.66 times that of hemoglobin, ratio of hydrochloric acid and acetone was 3%, the extraction time was 42.00 min, the maximum extraction rate of heme was 0.225 g/100 mL.
2 Initial structure identification of SOD and heme
Analyzed extracted SOD samples by UV spectrum, the maximum absorption peak is 265 nm, the result was consistent with those reported; compared the spectrogram of standard heme and samples heme by UV spectrum, Infrared spectrum and high performance liquid chromatography, the extraction sample was proved to be heme. Under the choosen mobile phase(methanol:water was 80:20) and equipment condition, detection limits, precision and stability was good, purity of heme was 72%.
3 Storage stability of SOD and heme
Freezing storage was conducive to the stability of SOD and heme in storage. In freezing storage, chill storage, room temperature and dark storage, room temperature in daylight storage, the relatively activity of SOD were 90%,85%,82%and 80.25%after 20 h. In freezing storage, chill storage, room temperature and dark storage, room temperature in daylight storage, the relatively contents of heme were 91.58%,88.61%,83.46% and 81.75% after 9 weeks.
4 Antioxidant activity and effects on mouse-borne tumor sp2/0 cell apoptosis of SOD
The antioxidant activity of SOD was detected in vitro, using Vc as the reference standard, the results showed that SOD had good antioxidant activity, but lower than that of Vc.
Observation of cell morphology is the basic method of apoptosis study. It was indicated that mouse-borne tumor SP2/0 cell grew well without SOD, showing round; mouse-borne tumor SP2/0 cell was significant nuclear condensation, fragmentation, showing the apoptotic morphology with different SOD concentration after a while, and with the increasing of the concentration, the extent of nuclear condensation and fragmentation increased. SOD can inhibit mouse-borne tumor SP2/0 cell proliferation to some extent in vitro, and showed time-effect and dose-effect relationship, SOD could induce mouse-borne tumor SP2/0 cell apoptosis.
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