复方促排铅功能制剂的组分优化及其生物活性研究
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摘要
本课题是国家“十五”食品安全重大科技专项——《食品安全关键技术应用的综合示范》的子课题暨黑龙江省科技攻关重点项目——《食品驱铅关键技术对铅中毒改善的研究与应用》(编号:GB04C109-01)、大庆科技局课题——《基于CAC标准的大豆分离蛋白含量、氮溶指数等优化工艺及中试研究》(编号:SGG2008-037)和大庆高新开发区创新基金——《大豆抗氧化肽提纯工艺的优化及在促排铅功能制剂中协同效果的研究》(编号:DQGX08YF027)的部分研究内容。
本文主要针对复方促排铅功能制剂的组分优化及其生物学活性展开研究。首先,对促排铅作用机理进行阐述,并对国内外促排铅药物和保健品现状进行对比分析;通过对高产蛋白酶的枯草芽孢杆菌基因工程菌的构建,对具有协同促排铅功效的大豆抗氧化肽进行了制备、分离和纯化的研究;再对药食同源的天然原料进行了单因素机器最佳剂量的筛选;利用数学统计方法创建了优化的数学模型,确立了复方促排铅功能制剂的优化组方,模糊数学评价其功效性;在此基础上,动物试验验证了该功能制剂促排铅的有效性和补充微量元素的可行性;最后结合对试验动物机体抗氧化损伤及修复的试验,综合体现和证实了该复方促排铅功能制剂的功效。
Lead poison, which can influence the function and life of erythrocyte, lead to the derangement of begetative nerve and hurt new protein composition and cranial nerve of key function for forming long-lasting memory, damage the function of renal tubule, and so on. Nowadays, there are two major clinical and health care remedial ways for curing lead poisoning. The experiment will develop and use homologous material of natural medicine and food to study functional compound recipe liquid to eliminating lead, which is of the more important preventing significance for people who have a certain lead accumulation in body, while not reaching poisoning content. Basing on studying preparation and purification of bean antioxidant peptide, the study is mainly aiming at the efficacy factor of compound functional oral liquid to establish mathematical model, evaluate economic character and fuzzily evaluate its efficacy, which provides the more scientific theoretical support in nutrition and healthcare. Researching content is as followed:
1. Clone and expression of antioxidant peptide pET-22b(+)-apr recombinant strain of using efficacy enzymolysis soybean protein. Bacillus subtilis protease apr was successfully cloned by PCR method and recombinant expression plasmid pET-22b (+)-apr was constructed and expressed in E.coli BL21(DE3), which full-length of gene sequence was 1509bp, 503 amino acid being coded, protein molecular weight being 55 ku, which the consistency of sequencing results was 100% with implementation sequence. Expression protein existed in inclusion body at 37℃.
2. The research of separating soybean antioxidant peptide with gel filtration chromatography. Using sephadex G-25 gel filtration chromatography to separate soybean hydrolysates, Four kinds of peptide components were obtained: SP1 in 78266Da above, SP2 of the range of 13240 ~ 78266Da, SP3 of the range of 1439 ~ 1067Da, SP4 of 156 ~ 1439Da. The anti-oxidation activity of these four kinds of peptide components was measured. The results showed that the antioxidant activity of SP4 was strongest. Through the amino acid composition analysis, peptide SP4 was composed of 16 kinds of amino acids. SP4 contained three peptide peaks with reversed-phase HPLC analysis.
3. The research of functional compound recipe liquid to eliminating lead design experiment and exterior-body functional appraisal. Through simulating stomach condition in exterior-body, the better ratio range and different dose chelated effect of every efficient elements were determined previously and every efficient elements’chelated effect in various doses were compared, then five factors A、B、C、D、E being obvious effect were studied. With penta-factorial quadratic orthogonal experimental design, the research combined the economic character of compound eliminating lead oral liquid and fuzzily appraised five factors of the oral liquid edible performance to determine the best combination: Gly being 1.46g, mixed extracted solution of Chinese medicine being 1.09mL, soybean antioxidant peptide being 0.94g, Zn being 0.33g and Ca being 0.17g.
4. Study on the eliminative functional compound recipe liquid to eliminating lead used to eliminate lead. 120“Kunming”rats being 18～20g were divided into 6 groups according to weight: blank group(BD), negative group(ND), positive group(PD) and high group (HD,6.67mg/kg.bw.d ),middle group (MD,3.33mg/kg.bw.d and low group(LD,1.67mg/kg.bw.d three dose groups). Except the BD, others groups drunk 0.24% Pb(AC)2, filled stomach with experimental liquid and DSMA. To 20d, blood were collected from tail once; to 40d, limosis 24h, liver, thighbone and blood were collected to determine the content of microelement: Pb2+, Ca2+, Zn2+. The results were as followed: lead content difference in plasma was extremely significant comparing the three dose containing eliminative lead compound oral liquid with BD, ND and PD(P<0.01); the diversity of lead content in liver and bone was not significant comparing MD with BD; The difference of HD bone lead content was not significant with PD. Eliminating lead compound oral liquid can supplement the good microelement Ca2+ and Zn2+ in body. The results proved that eliminating lead compound oral liquid had extremely difference with BD, ND and PD(P<0.01,P<0.05).
5. Study on the antioxidative function of functional compound recipe liquid to eliminating lead. 75“Kunming”rats being 18～20g are divided into 5 groups according to weight: BD、MoD、HD ( 6.67mg/kg.bw.d )、MD ( 3.33mg/kg.bw.d ) and LD (1.67mg/kg.bw.d). Freely drunk, filled stomach with experimental liquid, to 4 weeks end and limosis 10h, then the SOD and GSH-PX activity in plasma and MD content in plasma and liver were determined. The result were as followed: three dose groups of eliminating lead drink took on strong antioxidative function, MDA content in plasma and liver being significantly decreased(P<0.01), SOD and GSH-PX activity being significantly increased (P<0.01).
本文主要针对复方促排铅功能制剂的组分优化及其生物学活性展开研究。首先,对促排铅作用机理进行阐述,并对国内外促排铅药物和保健品现状进行对比分析;通过对高产蛋白酶的枯草芽孢杆菌基因工程菌的构建,对具有协同促排铅功效的大豆抗氧化肽进行了制备、分离和纯化的研究;再对药食同源的天然原料进行了单因素机器最佳剂量的筛选;利用数学统计方法创建了优化的数学模型,确立了复方促排铅功能制剂的优化组方,模糊数学评价其功效性;在此基础上,动物试验验证了该功能制剂促排铅的有效性和补充微量元素的可行性;最后结合对试验动物机体抗氧化损伤及修复的试验,综合体现和证实了该复方促排铅功能制剂的功效。
Lead poison, which can influence the function and life of erythrocyte, lead to the derangement of begetative nerve and hurt new protein composition and cranial nerve of key function for forming long-lasting memory, damage the function of renal tubule, and so on. Nowadays, there are two major clinical and health care remedial ways for curing lead poisoning. The experiment will develop and use homologous material of natural medicine and food to study functional compound recipe liquid to eliminating lead, which is of the more important preventing significance for people who have a certain lead accumulation in body, while not reaching poisoning content. Basing on studying preparation and purification of bean antioxidant peptide, the study is mainly aiming at the efficacy factor of compound functional oral liquid to establish mathematical model, evaluate economic character and fuzzily evaluate its efficacy, which provides the more scientific theoretical support in nutrition and healthcare. Researching content is as followed:
1. Clone and expression of antioxidant peptide pET-22b(+)-apr recombinant strain of using efficacy enzymolysis soybean protein. Bacillus subtilis protease apr was successfully cloned by PCR method and recombinant expression plasmid pET-22b (+)-apr was constructed and expressed in E.coli BL21(DE3), which full-length of gene sequence was 1509bp, 503 amino acid being coded, protein molecular weight being 55 ku, which the consistency of sequencing results was 100% with implementation sequence. Expression protein existed in inclusion body at 37℃.
2. The research of separating soybean antioxidant peptide with gel filtration chromatography. Using sephadex G-25 gel filtration chromatography to separate soybean hydrolysates, Four kinds of peptide components were obtained: SP1 in 78266Da above, SP2 of the range of 13240 ~ 78266Da, SP3 of the range of 1439 ~ 1067Da, SP4 of 156 ~ 1439Da. The anti-oxidation activity of these four kinds of peptide components was measured. The results showed that the antioxidant activity of SP4 was strongest. Through the amino acid composition analysis, peptide SP4 was composed of 16 kinds of amino acids. SP4 contained three peptide peaks with reversed-phase HPLC analysis.
3. The research of functional compound recipe liquid to eliminating lead design experiment and exterior-body functional appraisal. Through simulating stomach condition in exterior-body, the better ratio range and different dose chelated effect of every efficient elements were determined previously and every efficient elements’chelated effect in various doses were compared, then five factors A、B、C、D、E being obvious effect were studied. With penta-factorial quadratic orthogonal experimental design, the research combined the economic character of compound eliminating lead oral liquid and fuzzily appraised five factors of the oral liquid edible performance to determine the best combination: Gly being 1.46g, mixed extracted solution of Chinese medicine being 1.09mL, soybean antioxidant peptide being 0.94g, Zn being 0.33g and Ca being 0.17g.
4. Study on the eliminative functional compound recipe liquid to eliminating lead used to eliminate lead. 120“Kunming”rats being 18～20g were divided into 6 groups according to weight: blank group(BD), negative group(ND), positive group(PD) and high group (HD,6.67mg/kg.bw.d ),middle group (MD,3.33mg/kg.bw.d and low group(LD,1.67mg/kg.bw.d three dose groups). Except the BD, others groups drunk 0.24% Pb(AC)2, filled stomach with experimental liquid and DSMA. To 20d, blood were collected from tail once; to 40d, limosis 24h, liver, thighbone and blood were collected to determine the content of microelement: Pb2+, Ca2+, Zn2+. The results were as followed: lead content difference in plasma was extremely significant comparing the three dose containing eliminative lead compound oral liquid with BD, ND and PD(P<0.01); the diversity of lead content in liver and bone was not significant comparing MD with BD; The difference of HD bone lead content was not significant with PD. Eliminating lead compound oral liquid can supplement the good microelement Ca2+ and Zn2+ in body. The results proved that eliminating lead compound oral liquid had extremely difference with BD, ND and PD(P<0.01,P<0.05).
5. Study on the antioxidative function of functional compound recipe liquid to eliminating lead. 75“Kunming”rats being 18～20g are divided into 5 groups according to weight: BD、MoD、HD ( 6.67mg/kg.bw.d )、MD ( 3.33mg/kg.bw.d ) and LD (1.67mg/kg.bw.d). Freely drunk, filled stomach with experimental liquid, to 4 weeks end and limosis 10h, then the SOD and GSH-PX activity in plasma and MD content in plasma and liver were determined. The result were as followed: three dose groups of eliminating lead drink took on strong antioxidative function, MDA content in plasma and liver being significantly decreased(P<0.01), SOD and GSH-PX activity being significantly increased (P<0.01).
引文
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