食源三肽ACE抑制活性构效关系研究
摘要
由于食源性血管紧张素转化酶(ACE)抑制肽具有安全性高,无明显副作用,因而从不同的食品中提取ACE抑制肽成为了国内外研究的热点。从廉价的食物蛋白源中提取得到ACE抑制活性肽对于食物蛋白源的综合利用有着很重要的学术和应用价值。本文对通过文献收集整理的140个ACE抑制三肽和z-scales描述值结合进行定量构效关系研究,建立了三肽ACE抑制活性预测模型,用已知的三肽抑制活性值对模型进行了检验。模拟人体消化酶系统,采用网络系统对多种食物蛋白进行虚拟水解,收集整理得到的三肽,根据定量构效关系建立的活性预测模型计算了这些三肽的ACE抑制活性,筛选出活性较高的三肽进行化学合成。测定合成肽的ACE抑制活性,与预测的活性对比,以期对活性预测模型做进一步的修正。为了探讨抑制机理,对活性最高的三肽做了抑制动力学研究,同时用高效液相色谱法(HPLC)验证了这个肽在食物蛋白水解液中确实存在。
本课题主要研究有一下几个方面:
食物蛋白源的ACE抑制三肽定量构效关系的建立:筛选了140个已知其抑制活性的三肽,确定其已知的IC_(50)值为自变量,采用z-scales描述值为因变量,进行分析,通过偏最小二乘法建立三肽定量构效模型,并采用已知抑制ACE活性的三肽进行检验。
食源性三肽库的建立:总结了多种食物蛋白质,采用网络软件虚拟水解这些食物蛋白质,筛选山三肽,并根据(1)所构建出的QSAR模型预测其IC_(50)值。
具有高ACE抑制活性三肽的合成:根据模型预测的IC_(50)值,选取活性较高的三肽化学合成,然后测定其实际IC_(50)值。对实际测定抑制活性最高的三肽做抑制动力学研究,以便了解其抑制机理,同时评估这个三肽体外抗消化酶水解的能力。
用合成的三肽为标准物质,用HPLC法在食物蛋白质水解物中寻找该种ACE抑制三肽,来验证其是否存在于水解液中。
通过实验研究获得了以下结果和结论:
QSAR模型:回归方程y=1.727584+0.060348x_1+0.055900x_2+0.106603x_3-0.041317x_4-0.060064x_5+0.151157x_6+0.070841x_7-0.145287x_8-0.071214x_9,N=140,R=0.7403,而交互检验(cross validation,CV)系数为0.5472,Rcv~2＞0.5,说明相应变量建立的模型具有较好的预测能力。QSAR研究结果表明,ACE抑制三肽C末端起第三位氨基酸残基的立体性状大小指数Z2(x8)对活性的影响最大,与三肽的ACE抑制活性呈负相关,当该位氨基酸残基的立体形状小而疏水性大时,满足这些条件的氨基酸主要为Phe、Trp、Leu、Ile、Val等氨基酸。第二影响因素是C端起第二位氨基酸的电性参数Z3(x6),电性参数越大,活性越强,如Ala、Gly、Lys和Val等氨基酸。C端氨基酸,主要决定于疏水性和侧链的体积,疏水性和侧链氨基酸体积大的氨基酸有利于提高三肽的活性。
以胃蛋白酶和胰蛋白酶为水解酶,虚拟水解多种食源性蛋白为多肽,筛选出的三肽,计算出预测值,并筛选出了IRL、VVF和LRL三个抑制活性高的三肽,其对应的IC_(50)预测值分别为2.44μmol/L、6.09μmol/L、2.79μmol/L。而实际测定这三个肽的IC_(50)分别为20.3μmol/L,14.36μmol/L和3.42μmol/L,其中LRL实际测定值和预测值非常接近。
抑制动力学研究表明,LRL对ACE的抑制属于竞争性抑制。即LRL结合在ACE的活性中心,其抑制常数Ki为2.81μmol/L。在体外用胃蛋白酶和胰蛋白酶处理后的LRL抑制活性分别为96.18%和93.96%,和处理前的96.28%相比较,没有太大的变化,预计此肽可以用于动物实验而能够保证在动物体内的ACE抑制活性。
高效相色谱分析结果表明,活性最高的三肽LRL在油菜蛋白水解液中存在。
Antiotensin-I Converting Enzyme(ACE) inhibitory peptides of food origin have been received increasing interests because of their higher safety.Isolation and characteristic of ACE inhibitory peptides from foodstuff has draw considerable attention of scientists who are interested in the relationship of food components and health factors.In this study,model of QSAR was proposed for 140 tri-peptide inhibitors of ACE based on z-scales.Test the model with the known ACEI tri-peptides IC_(50) value.And then simulated human digestive system,using network software to dummy hydrolyze the many kinds of food derived protein,choose out the tri-peptides and then using the QSAR model to predict the IC_(50) values.Composed the highest ACE inhibitory activity tri-peptides,tested the practical IC_(50) values and compared with the predicted values,which could amend the QSAR model.For the sake of discussing the inhibitory mechanism,we did inhibitory dynamics research.After that the tri-peptides with highest ACE inhibitory activity was determined in the practical food hydrolysate by HPLC using synthetic peptides as reference.
The approaches to food ACE inhibitors by this paper can be concluded as follows:
Construction of quantitative structure-activity relationships(QSAR) model of ACE inhibitory tri-peptides derived from food-protein:140 ACEI tri-peptides were chosen from literature.The structure of these chosen tri-peptides were numeralized using z-scales descriptors and partial least square methods(PLSM) for parameterizing the structural variability of ACEI tri-peptides,then developed a QSAR model.The model was tested by known tri-peptides.
Establishment of the library of food origin tri-peptides:summing up many kinds of food-derived proteins,using network software to virtually hydrolyze the protein,chose out the tri-peptides and then through the QSAR model to predict the IC_(50) values.
Composed the higher inhibitory tri-peptides:Chose the higher ACE inhibitory tri-peptides to compose through the QSAR model predicted IC_(50) values.Then their actual IC_(50) values were measured,chose out reasonable tri-peptide,researched the inhibitory mechanism and evaluated the digesting enzyme resistance of these tri-peptides.
Used the synthetical tri-peptides as standard substance,trace of tri-peptide with highest ACE inhibitory activity by HPLC in food hydrolytes.
The results of this paper summarized as follows.
QSAR model y=1.727584+0.060348x_1+0.055900x_2+0.106603x_3-0.041317x_4-0.060064x_5+ 0.151157x_6+0.070841x_7-0.145287x_8-0.071214x_9 was obtained using partial least square method (PLSM),and the regression analysis methods,the multiple correlation R is 0.7403,and the cross validation RCV~2 is 0.5472,it was also observed that the size of three-dimensional characters index Z_2(x8) of the antepenultimate amino acid residue from the C-terminal,which is the most important factor affecting the ACE inhibitory,and is negatively correlated to the activity,the hydrophobicity of tri-peptide positively correlated to activity.Such amino acid as Phe、Trp、Leu、Ile、Val.The second important factor is Electric parameters Z_3(x_6) of the penultimate amino acid residue from the C-terminal,which is positively correlated to activity,Such amino acid as Ala、Gly、Lys and Val The C-terminal amino acid is determined the hydrophobic ability and the volume of side chain, which are positively correlatively correlated to ability.
Used pepsin and trypsin as hydrolyzed enzyme,virtually hydrolyzed many food derived protein, chose out tri-peptides and figured out the IC_(50) value,the highest inhibitory IC_(50) were IRL、VVF&LRL,The predicted IC_(50) values 2.44μmol/L、6.09μmol/L、2.79μmol/L.the actual IC_(50) were 20.3μmol/L,14.36μmol/L&3.42μmol/L,respectively.The predict value resembled as the actual value of LRL.
The inhibitory mechanism research showed that:LRL inhibitory type was competition inhibitor when it effected on ACE,which showed that LRL integrated in the activity center of ACE. Inhibitory constant Ki was 2.81μmol/L.Used pepsin and trypsin as hydrolyzed enzyme hydrolyzed LRL in vitro,the inhibitory rate were 96.18%and 93.96%respectively,there was not noticeable difference compared with no hydrolization 96.28%.So we forecasted that LRL could be used in animal experiment and kept the activity.
HPLC result showed that LRL existed in the water extracting substance of rapeseed protein.
本课题主要研究有一下几个方面:
食物蛋白源的ACE抑制三肽定量构效关系的建立:筛选了140个已知其抑制活性的三肽,确定其已知的IC_(50)值为自变量,采用z-scales描述值为因变量,进行分析,通过偏最小二乘法建立三肽定量构效模型,并采用已知抑制ACE活性的三肽进行检验。
食源性三肽库的建立:总结了多种食物蛋白质,采用网络软件虚拟水解这些食物蛋白质,筛选山三肽,并根据(1)所构建出的QSAR模型预测其IC_(50)值。
具有高ACE抑制活性三肽的合成:根据模型预测的IC_(50)值,选取活性较高的三肽化学合成,然后测定其实际IC_(50)值。对实际测定抑制活性最高的三肽做抑制动力学研究,以便了解其抑制机理,同时评估这个三肽体外抗消化酶水解的能力。
用合成的三肽为标准物质,用HPLC法在食物蛋白质水解物中寻找该种ACE抑制三肽,来验证其是否存在于水解液中。
通过实验研究获得了以下结果和结论:
QSAR模型:回归方程y=1.727584+0.060348x_1+0.055900x_2+0.106603x_3-0.041317x_4-0.060064x_5+0.151157x_6+0.070841x_7-0.145287x_8-0.071214x_9,N=140,R=0.7403,而交互检验(cross validation,CV)系数为0.5472,Rcv~2＞0.5,说明相应变量建立的模型具有较好的预测能力。QSAR研究结果表明,ACE抑制三肽C末端起第三位氨基酸残基的立体性状大小指数Z2(x8)对活性的影响最大,与三肽的ACE抑制活性呈负相关,当该位氨基酸残基的立体形状小而疏水性大时,满足这些条件的氨基酸主要为Phe、Trp、Leu、Ile、Val等氨基酸。第二影响因素是C端起第二位氨基酸的电性参数Z3(x6),电性参数越大,活性越强,如Ala、Gly、Lys和Val等氨基酸。C端氨基酸,主要决定于疏水性和侧链的体积,疏水性和侧链氨基酸体积大的氨基酸有利于提高三肽的活性。
以胃蛋白酶和胰蛋白酶为水解酶,虚拟水解多种食源性蛋白为多肽,筛选出的三肽,计算出预测值,并筛选出了IRL、VVF和LRL三个抑制活性高的三肽,其对应的IC_(50)预测值分别为2.44μmol/L、6.09μmol/L、2.79μmol/L。而实际测定这三个肽的IC_(50)分别为20.3μmol/L,14.36μmol/L和3.42μmol/L,其中LRL实际测定值和预测值非常接近。
抑制动力学研究表明,LRL对ACE的抑制属于竞争性抑制。即LRL结合在ACE的活性中心,其抑制常数Ki为2.81μmol/L。在体外用胃蛋白酶和胰蛋白酶处理后的LRL抑制活性分别为96.18%和93.96%,和处理前的96.28%相比较,没有太大的变化,预计此肽可以用于动物实验而能够保证在动物体内的ACE抑制活性。
高效相色谱分析结果表明,活性最高的三肽LRL在油菜蛋白水解液中存在。
Antiotensin-I Converting Enzyme(ACE) inhibitory peptides of food origin have been received increasing interests because of their higher safety.Isolation and characteristic of ACE inhibitory peptides from foodstuff has draw considerable attention of scientists who are interested in the relationship of food components and health factors.In this study,model of QSAR was proposed for 140 tri-peptide inhibitors of ACE based on z-scales.Test the model with the known ACEI tri-peptides IC_(50) value.And then simulated human digestive system,using network software to dummy hydrolyze the many kinds of food derived protein,choose out the tri-peptides and then using the QSAR model to predict the IC_(50) values.Composed the highest ACE inhibitory activity tri-peptides,tested the practical IC_(50) values and compared with the predicted values,which could amend the QSAR model.For the sake of discussing the inhibitory mechanism,we did inhibitory dynamics research.After that the tri-peptides with highest ACE inhibitory activity was determined in the practical food hydrolysate by HPLC using synthetic peptides as reference.
The approaches to food ACE inhibitors by this paper can be concluded as follows:
Construction of quantitative structure-activity relationships(QSAR) model of ACE inhibitory tri-peptides derived from food-protein:140 ACEI tri-peptides were chosen from literature.The structure of these chosen tri-peptides were numeralized using z-scales descriptors and partial least square methods(PLSM) for parameterizing the structural variability of ACEI tri-peptides,then developed a QSAR model.The model was tested by known tri-peptides.
Establishment of the library of food origin tri-peptides:summing up many kinds of food-derived proteins,using network software to virtually hydrolyze the protein,chose out the tri-peptides and then through the QSAR model to predict the IC_(50) values.
Composed the higher inhibitory tri-peptides:Chose the higher ACE inhibitory tri-peptides to compose through the QSAR model predicted IC_(50) values.Then their actual IC_(50) values were measured,chose out reasonable tri-peptide,researched the inhibitory mechanism and evaluated the digesting enzyme resistance of these tri-peptides.
Used the synthetical tri-peptides as standard substance,trace of tri-peptide with highest ACE inhibitory activity by HPLC in food hydrolytes.
The results of this paper summarized as follows.
QSAR model y=1.727584+0.060348x_1+0.055900x_2+0.106603x_3-0.041317x_4-0.060064x_5+ 0.151157x_6+0.070841x_7-0.145287x_8-0.071214x_9 was obtained using partial least square method (PLSM),and the regression analysis methods,the multiple correlation R is 0.7403,and the cross validation RCV~2 is 0.5472,it was also observed that the size of three-dimensional characters index Z_2(x8) of the antepenultimate amino acid residue from the C-terminal,which is the most important factor affecting the ACE inhibitory,and is negatively correlated to the activity,the hydrophobicity of tri-peptide positively correlated to activity.Such amino acid as Phe、Trp、Leu、Ile、Val.The second important factor is Electric parameters Z_3(x_6) of the penultimate amino acid residue from the C-terminal,which is positively correlated to activity,Such amino acid as Ala、Gly、Lys and Val The C-terminal amino acid is determined the hydrophobic ability and the volume of side chain, which are positively correlatively correlated to ability.
Used pepsin and trypsin as hydrolyzed enzyme,virtually hydrolyzed many food derived protein, chose out tri-peptides and figured out the IC_(50) value,the highest inhibitory IC_(50) were IRL、VVF&LRL,The predicted IC_(50) values 2.44μmol/L、6.09μmol/L、2.79μmol/L.the actual IC_(50) were 20.3μmol/L,14.36μmol/L&3.42μmol/L,respectively.The predict value resembled as the actual value of LRL.
The inhibitory mechanism research showed that:LRL inhibitory type was competition inhibitor when it effected on ACE,which showed that LRL integrated in the activity center of ACE. Inhibitory constant Ki was 2.81μmol/L.Used pepsin and trypsin as hydrolyzed enzyme hydrolyzed LRL in vitro,the inhibitory rate were 96.18%and 93.96%respectively,there was not noticeable difference compared with no hydrolization 96.28%.So we forecasted that LRL could be used in animal experiment and kept the activity.
HPLC result showed that LRL existed in the water extracting substance of rapeseed protein.
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