菜籽肽的生物活性与构象研究
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摘要
人类摄食的蛋白质经消化道酶作用后,主要是以低聚肽形式被吸收,某些低聚肽不仅能提供人体生长、发育所需的营养物质,还具有防病治病、调节人体生理机能的功效。菜籽肽的氨基酸含量多且平衡良好,并含有多种生物活性。菜籽肽已成为目前植物源活性肽研究领域的新热点。
本研究通过柱层析系统、液质联用、傅立叶-拉曼光谱、红外光谱、分子模拟等近代最新技术,系统地研究了菜籽肽的理化性质、分离纯化、生物活性及空间构象,结果如下:
采用Alcalase和Flavourzyme分步水解得到菜籽肽并对其理化性质进行研究,通过葡聚糖凝胶层析获得了RSP-1、RSP-2、RSP-3和RSP-4共4个组分。对菜籽肽各级分的清除自由基作用和抑制血管紧张素转化酶(ACE)作用表明,RSP-4的清除ABTS自由基作用(TEAC值=0.24)和抑制ACE作用(IC50=0.19 mg/mL)最强,而其清除DPPH作用(IC50=0.18 mg/mL)则仅低于RSP-1(IC50=0.125 mg/mL)和RSP-2(IC50=0.125 mg/mL)。
采用液质联用、傅立叶-拉曼光谱、红外光谱、分子模拟等技术对RSP-4构象进行了深入研究与分析。通过液质联用技术确定RSP-4的分子量为977Da,结合数据库检索可推测其氨基酸序列为PFDSYFVC。拉曼光谱和红外光谱解析不但验证了质谱所得的氨基酸组成信息,拉曼光谱更进一步表明其二级空间结构为β-转角。通过分子模拟技术对RSP-4三维空间结构进行预测。按照RSP-4序列进行多肽合成,并对其生物活性进行进一步研究。
该研究对菜籽肽的生物活性和构象进行了系统深入的研究,其结果为天然活性肽在生物医药中的应用提供了科学依据和理论基础,同时为植物源活性肽的开发与应用提供了新的思路、借鉴。
After digestive enzymes hydrolyzed, protein is absorbed in the form of oligopeptide. Some oligopeptides not only provide nutrients which human growth and development needed, but also prevent and control disease, regulate physiological functions. Rapeseed peptide (RSP) is rich in amino acid, the amino acid composition of which is well-balanced. Besides,it contains a variety of bioactivities. Deep processing and utilization of RSP has become a new hot research in plant peptide field.
In this study, the physicochemical properties, separation and purification, bioactivity and conformation of RSP were systematically studied, using the latest modern technology including column chromatography system, liquid chromatography-mass spectrometry (LC-MS), FT - Raman spectroscopy, infrared spectroscopy and molecular simulation. The main results are shown as follows: A research on rapeseed protein was sequentially hydrolyzed with alcalase and flavourzyme were performed. Based on this, the physicochemical properties of RSP were studied. RSP was separated by Sephadex chromatography into four components: RSP-1,RSP-2 , RSP-3 and RSP-4.
The bioactivities of crude rapeseed peptide and its fractions were investigated, including the free radical scavenging and ACE inhibition. In all fractions, the RSP-4’s ABTS free radical scavenging capacity (TEAC value = 0.24) and ACE inhibition capacity (IC50 = 0.19 mg/mL) were strongest, whereas the DPPH free radical scavenging activity of RSP-4 (IC50 = 0.18 mg/mL) was slightly lower than that of RSP-1 (IC50 = 0.125 mg/mL) and RSP-2 (IC50 = 0.125 mg/ mL).
The conformation of RSP-4 was first investigated by modern technique including LC-MS, FT - Raman spectroscopy, infrared spectroscopy and molecular simulation. LC-MS determined the molecular weight of RSP-4 was 977Da, combining with database search deduced its amino acid sequence was PFDSYFVC. FT - Raman spectroscopy and infrared spectroscopy verified the amino acid composition information obtained from LC-MS. FT - Raman spectra showed that the secondary structure of RSP-4 wasβangle. Molecular simulation predicted the three-dimensional structure of RSP-4. Peptide was synthesized in accordance with the RSP-4 sequence and determined its bioactivity.
This study in-depth investigated the bioactivity and conformation of RSP. These results not only provide a scientific and theoretical basis for natural peptide in biomedical application, but also give new ideas to the source of development and application of plant peptide.
本研究通过柱层析系统、液质联用、傅立叶-拉曼光谱、红外光谱、分子模拟等近代最新技术,系统地研究了菜籽肽的理化性质、分离纯化、生物活性及空间构象,结果如下:
采用Alcalase和Flavourzyme分步水解得到菜籽肽并对其理化性质进行研究,通过葡聚糖凝胶层析获得了RSP-1、RSP-2、RSP-3和RSP-4共4个组分。对菜籽肽各级分的清除自由基作用和抑制血管紧张素转化酶(ACE)作用表明,RSP-4的清除ABTS自由基作用(TEAC值=0.24)和抑制ACE作用(IC50=0.19 mg/mL)最强,而其清除DPPH作用(IC50=0.18 mg/mL)则仅低于RSP-1(IC50=0.125 mg/mL)和RSP-2(IC50=0.125 mg/mL)。
采用液质联用、傅立叶-拉曼光谱、红外光谱、分子模拟等技术对RSP-4构象进行了深入研究与分析。通过液质联用技术确定RSP-4的分子量为977Da,结合数据库检索可推测其氨基酸序列为PFDSYFVC。拉曼光谱和红外光谱解析不但验证了质谱所得的氨基酸组成信息,拉曼光谱更进一步表明其二级空间结构为β-转角。通过分子模拟技术对RSP-4三维空间结构进行预测。按照RSP-4序列进行多肽合成,并对其生物活性进行进一步研究。
该研究对菜籽肽的生物活性和构象进行了系统深入的研究,其结果为天然活性肽在生物医药中的应用提供了科学依据和理论基础,同时为植物源活性肽的开发与应用提供了新的思路、借鉴。
After digestive enzymes hydrolyzed, protein is absorbed in the form of oligopeptide. Some oligopeptides not only provide nutrients which human growth and development needed, but also prevent and control disease, regulate physiological functions. Rapeseed peptide (RSP) is rich in amino acid, the amino acid composition of which is well-balanced. Besides,it contains a variety of bioactivities. Deep processing and utilization of RSP has become a new hot research in plant peptide field.
In this study, the physicochemical properties, separation and purification, bioactivity and conformation of RSP were systematically studied, using the latest modern technology including column chromatography system, liquid chromatography-mass spectrometry (LC-MS), FT - Raman spectroscopy, infrared spectroscopy and molecular simulation. The main results are shown as follows: A research on rapeseed protein was sequentially hydrolyzed with alcalase and flavourzyme were performed. Based on this, the physicochemical properties of RSP were studied. RSP was separated by Sephadex chromatography into four components: RSP-1,RSP-2 , RSP-3 and RSP-4.
The bioactivities of crude rapeseed peptide and its fractions were investigated, including the free radical scavenging and ACE inhibition. In all fractions, the RSP-4’s ABTS free radical scavenging capacity (TEAC value = 0.24) and ACE inhibition capacity (IC50 = 0.19 mg/mL) were strongest, whereas the DPPH free radical scavenging activity of RSP-4 (IC50 = 0.18 mg/mL) was slightly lower than that of RSP-1 (IC50 = 0.125 mg/mL) and RSP-2 (IC50 = 0.125 mg/ mL).
The conformation of RSP-4 was first investigated by modern technique including LC-MS, FT - Raman spectroscopy, infrared spectroscopy and molecular simulation. LC-MS determined the molecular weight of RSP-4 was 977Da, combining with database search deduced its amino acid sequence was PFDSYFVC. FT - Raman spectroscopy and infrared spectroscopy verified the amino acid composition information obtained from LC-MS. FT - Raman spectra showed that the secondary structure of RSP-4 wasβangle. Molecular simulation predicted the three-dimensional structure of RSP-4. Peptide was synthesized in accordance with the RSP-4 sequence and determined its bioactivity.
This study in-depth investigated the bioactivity and conformation of RSP. These results not only provide a scientific and theoretical basis for natural peptide in biomedical application, but also give new ideas to the source of development and application of plant peptide.
引文
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