【作者】 邹平；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2014， 博士

【摘要】 高血压已成为最常见的严重的全球慢性病的健康问题之一,会同时引起或加重大脑、心血管、肾脏和肝脏的损害。另一方面,人体内的脂质氧化促进病变和衰老等问题,而且血压和脂质氧化有着密切的联系。因此,通过改变饮食习惯和添加天然食品中的功能性成分来辅助降血压、清除自由基,有着重要意义。菜粕是我国的主要农作物副产品之一,其蛋白含量高,氨基酸组成是比较平衡,易于消化吸收被机体利用,但目前应用方式还比较简单。本研究以菜籽蛋白为研究对象,结合生物信息学、QSAR和分子对接等方法,筛选了抗氧化肽和ACE抑制肽,并进行了活性和机理研究。本文分别用Thermolysin-Pepsin,Thermo lysin-Trypsin组合水解Napin和Cruciferin得到含有数百种菜籽蛋白肽的多肽库,而后建立了从菜籽蛋白水解液中快速鉴别抗氧化肽的方法。结合NANO-Q-TOF质谱技术确证了水解液中存在4条抗氧化肽,分别为HL, IR, RW和VY。通过源蛋白序列比对,获悉了这些多肽的酶切位点图,此方法高效快速准确。通过QSAR预测由计算机辅助水解菜籽蛋白得到多肽的IC50,并结合NANO-Q-TOF质谱技术找到了水解液中存在6条ACE抑制肽,再利用分子对接方法评价其活性,筛选获得了一条高活性的ACE抑制肽GRD,其预测活性为7.54gM,且目前尚未有文献报道,是一条新发现的ACE抑制肽,其来源蛋白是一种Napin蛋白(oleosin-like protein, NCBI中ID:ABD14345),可由Pepsin水解获得。利用CDOCKER方法对4-6肽进行了系统性筛选,结果表明,四肽IYKY、 IYRT和IRQQ;五肽QQEEP、IRQQL和IYDVR以及六肽VRGYKK、YKKILV和RDMHQK是最可能的高活性ACE抑制肽通过自发性高血压大鼠(SHR)模型,研究了多肽在体内的降血压作用,并揭示FL和VSV对ACE抑制的机理。多肽FL和VSV对SHR大鼠的SBP均有降低效果,其中VSV高剂组在4h时下降22.8mmHg,长期给药可降低23.6mmHgo这表明,VSV有望作为辅助剂降血压的功能性食品在未来的治疗中使用。分子对接结果显示：多肽-蛋白活性区域-水分子之间存在复杂的氢键、分子间作用力等作用,从而形成竞争性抑制。测定了SHR大鼠血清、肝脏和脑组织的SOD、CAT活性以及MDA、GSH含量,结果表明FL和VSV具有一定的抗氧化能力。本研究的结果为开发利用菜籽蛋白资源作为功能性食品原料,提高菜籽附加值提高了研究依据,且建立了快速筛选生物活性肽的方法,为生物活性肽研究提供了新的思路。更多还原

【Abstract】 Hypertension has become one of the most common serious worldwide chronic health problems and it will also cause or aggravate brain, cardiovascular, kidney and liver damage. On the other hand, blood pressure and lipid oxidation are closely linked, and the lipid oxidation of human body promotes disease and aging issues. Therefore, reducing blood pressure and scavenging free radical by changing diet habits and adding of natural functional ingredients in foods are of great significance.Rapeseed meal is one of the major crop by-products,and it has high content of protein and balanced amino acid composition, what’s more, it is easy to digest and absorpt by the body.However, the application of rapeseed meal is still relatively simple. In this research, antioxidant and ACE inhibitory peptides from rapeseed protein were screened by bioinformatics, QSAR and molecular docking method, at the same time, their activity and mechanism were also studied.In this paper, hundreds of peptides from Napin and Cruciferin were got by in-silico hydrolysis by combinat or respective use of Thermolysin-Pepsin or Thermolysin-Trypsin, and then we established an approach for rapid identification of antioxidant peptides from rapeseed protein hydrolyzate by combined use with NANO-Q-TOF mass spectrometry. Four peptides(HL, IR, RW and VY) were found in hydrolyzate, finally the cleavage sites map was built by sequence alignment. This method was fast, efficient and accurate.The IC50of peptides obtained by in-silico hydrolysisfrom rapeseed protein was predicted by QSARmodel.Then six ACE inhibitory peptides were found in hydrolyzate by NANO-Q-TOF mass spectrometry.A peptide (sequenced GRD) with high ACE inhibitory activity was screened by molecular docking method and its predicted IC50was7.54μM. GRD has not been reported in the literature, so it is a newly discovered ACE inhibitory peptide. Its source protein is a kinf of Napin protein (oleosin-Iike protein, ID in NCBI:ABD14345), and it can be obtained by hydrolysis using Pepsin. CDOCKER method was used to screen peptide with4-6 amino acid residues, the results showed that tetrapeptide IYKY, IYRT and IRQQ; pentapeptide QQEEP, IRQQL and IYDVR and hexapeptide VRGYKK, YKKILV and RDMHQK are most likely highly active ACE inhibitory peptides.Spontaneously hypertensive rats (SHR) model was used to study the blood pressure lowering effect of peptides in vivo, and to reveal the ACE inhibitory mechanism of VSV and on FL. FL and VSV both had were SBP-lowering effect in SHR rats, and SBP of VSV high-dose group decreased by22.8mmHg during4h after administration, and23.6mmHg in long-term administration experiment respectively. This indicated that VSV was expected as a functional food or hypotensive agent in future treatment. Docking results showed that:hydrogen bonds and the role of intermolecular forces were formed among peptide, protein active region and water molecules, thereby the peptides created a competitive inhibition. Both FL and VSV promote activity of antioxidant enzymes, SOD and CAT, with increasing MDA level in brain and decreasing reduced GSH level in SHRs to a different extent after long-term oral administration. This suggested that FL and VSV can be as added as antioxidant in functional food.The results of this study provided basis for the development and utilization of resources rapeseed proteins as functional food ingredients to improve the added value. And the establishment of a rapid method for bioactive peptides screening gave a new thinking of bioactive peptides research.更多还原