1-脱氧野尻霉素对α-葡萄糖苷酶的抑制作用机制
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摘要
运用紫外光谱法、荧光光谱法和圆二色光谱法,研究桑叶提取物1-脱氧野尻霉素(1-deoxynojirimycin,DNJ)对α-葡萄糖苷酶的作用。结果发现:DNJ与α-葡萄糖苷酶反应的半抑制浓度为0.297μg/mL,作用类型为竞争型抑制;其与α-葡萄糖苷酶主要通过静电吸引力相互作用形成基态复合物,并使α-葡萄糖苷酶的内源荧光猝灭;DNJ与α-葡萄糖苷酶相互作用形成复合物的过程是熵驱动的吸热反应,静电吸引力是两者结合反应的主要驱动力。不同温度(273、298、310 K)条件下荧光猝灭常数(K_(sv))分别为1.48×10~4、1.29×10~4、1.12×10~4 L/mol。DNJ使α-葡萄糖苷酶的构象发生变化,且使其二级结构重新排列;诱导酶活性口袋关闭,不利于底物结合到活性位点,推测这可能是DNJ抑制α-葡萄糖苷酶活性从而降低血糖水平的机理。
The inhibitory effect of 1-deoxynojirimycin(DNJ) extracted from mulberry leaves on α-glycosidase was investigated by ultraviolet, ?uorescence and circular dichroism(CD) spectroscopy. As a result, the half maximum inhibitory concentration(IC_(50)) of DNJ α-glucosidase was 0.297 μg/mL. The type of inhibition was competitive. DNJ interacted withα-glucosidase through electrostatic attraction generating ground-state complexes and resulting in endogenous ?uorescence quenching of α-glucosidase. The formation process of DNJ-α-glucosidase complexes was an entropy-driven endothermic reaction. The interaction was mainly driven by electrostatic attraction forces. At different temperatures(273, 298 and 310 K),?uorescence quenching constants(K_(sv)) were 1.48 × 10~4, 1.29 × 10~4, and 1.12 × 10~4 L/mol, respectively. DNJ resulted in a conformational change of α-glucosidase and rearrangement of the secondary structure inducing closing of the enzyme active pocket and consequently inhibiting substrate binding to the active center. These ?ndings may explain the mechanism by which DNJ inhibited α-glucosidase activity and thus reduced blood glucose levels.
The inhibitory effect of 1-deoxynojirimycin(DNJ) extracted from mulberry leaves on α-glycosidase was investigated by ultraviolet, ?uorescence and circular dichroism(CD) spectroscopy. As a result, the half maximum inhibitory concentration(IC_(50)) of DNJ α-glucosidase was 0.297 μg/mL. The type of inhibition was competitive. DNJ interacted withα-glucosidase through electrostatic attraction generating ground-state complexes and resulting in endogenous ?uorescence quenching of α-glucosidase. The formation process of DNJ-α-glucosidase complexes was an entropy-driven endothermic reaction. The interaction was mainly driven by electrostatic attraction forces. At different temperatures(273, 298 and 310 K),?uorescence quenching constants(K_(sv)) were 1.48 × 10~4, 1.29 × 10~4, and 1.12 × 10~4 L/mol, respectively. DNJ resulted in a conformational change of α-glucosidase and rearrangement of the secondary structure inducing closing of the enzyme active pocket and consequently inhibiting substrate binding to the active center. These ?ndings may explain the mechanism by which DNJ inhibited α-glucosidase activity and thus reduced blood glucose levels.
引文
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[2]姚起鑫,亓竹青,王光等.槟榔碱改善2型糖尿病大鼠糖、脂代谢紊乱[J].中国药理学通报,2009,25(11):1477-1481.DOI:10.3321/j.issn:1001-1978.2009.11.020.
[3]徐小军,余国珍,胡礼勇.桑树各药用部位1-脱氧野尻霉素的含量考察[J].中药材,2010,33(1):28-30.
[4]国家药典委员会.中华人民共和国药典:一部[M].北京:中国医药科技出版社,2010:191.
[5]李时珍.本草纲目[M].北京:人民卫生出版社,2004:865-868.
[6]杨雨,欧阳臻,常钰,等.桑叶不同组分降血糖作用研究[J].食品科学,2007,28(8):454-456.DOI:10.3321/j.issn:1002-6630.2007.08.112.
[7]佐藤修二.桑叶提取物对大鼠小肠二糖类吸收的抑制作用[J].国外医学(中医中药分册),1999(4):54-58.
[8]金航.桑叶对二糖酶的抑制作用[J].国外医学(中医中药分册),1999(3):38-39.
[9]FANG M,HUANG A Q,ZHANG Y Q.The correlation between1-deoxynojirimycin content and alpha-glucosidase inhibitory activity in the bark ethanol extract from Ramulus mori[C]//International Conference on Biomedical Engineering and Biotechnology.Washington:IEEE Computer Society,2012:1795-1798.DOI:10.1109/iCBEB.2012.406.
[10]KIMURAM,CHEN F.Antihypelycemic effects of N-containing sugars derived from mulberry leaves is streptozocin-in-duced diabetic mice[J].Wakan Iyakugaku Zasshi,1995,12(3):214-219.
[11]HSIEH J F,LIN W J,HUANG K F,et al.Antioxidant activity and inhibition ofα-glucosidase by hydroxyl-functionalized 2-arylbenzo[b]furans[J].European Journal of Medicinal Chemistry,2015,93:443-451.
[12]尚禹东.α-糖苷酶抑制剂:银杏叶提取物的药效学研究[D].长春:长春中医药大学,2010:11-16.
[13]PATEL S S.Cerebrovascular complications of diabetes:alpha glucosidase inhibitor as potential therapy[J].Hormone&Metabolic Research,2015,48(2):83-91.DOI:10.1055/s-0035-1565181.
[14]刘里,杨晓丽,成飞翔.光谱法研究炎琥宁与牛血清白蛋白的相互作用[J].世界科学技术-中医药现代化,2015,17(5):1113-1118.DOI:10.11842/wst.2015.05.037.
[15]孙萍,李鸿丽,王修中.光谱法研究硝基苯与牛血清蛋白的相互作用[J].化学研究与应用,2014,26(6):804-808.DOI:10.3969/j.issn.1004-1656.2014.06.005.
[16]张秋菊.光谱法研究格列齐特、格列喹酮、硫酸粘杆菌素与牛血清白蛋白间的相互作用[D].保定:河北大学,2016:1-2.
[17]王永刚,王世伟,李云春,等.考马斯亮蓝G-250与牛血清白蛋白的相互作用研究[J].现代食品科技,2016,32(1):89-94.
[18]王晓云,任峻青,赵秀花,等.紫外光谱法研究7-羟基香豆素与溶菌酶的相互作用[J].贵阳中医学院学报,2013,35(4):293-295.DOI:10.3969/j.issn.1002-1108.2013.04.0155.
[19]ZHANG L H,LIU B S,LI Z Y,et al.Comparative studies on the interaction of cefixime with bovine serum albumin by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy[J].Spectroscopy Letters,2015,48(6):441-446.DOI:10.1080/00387010.2014.909493.
[20]秦宁波,李达翃,李占林,等.圆二色谱法在黄酮木脂素绝对构型测定中的应用进展[J].国际药学研究杂志,2015,42(6):794-801.DOI:10.13220/j.cnki.jipr.2015.06.014.
[21]SREERAMA N,VENYAMINOV S Y U,WOODY R W.Estimation of the number ofα-helical andβ-strand segments in proteins using circular dichroism spectroscopy[J].Protein Science,2010,8(2):370-380.
[22]DARDEN T,YORK D,PEDERSEN L.Particle mesh Ewald:an N?log(N)method for Ewald sums in large systems[J].The Journal of Chemical Physics,1993,98(12):10089-10092.DOI:10.1063/1.464397.
[23]刘家琴,王坤,闻春雷,等.荧光光谱法研究磺脲类降糖药物与牛血清白蛋白的相互作用[J].周口师范学院学报,2015,32(2):79-82.DOI:10.13450/j.cnki.jzknu.2015.02.022.
[24]PENG Xin,WANG Xiangchao,QI Wei,et al.Affinity of rosmarinic acid to human serum albumin and its effect on protein conformation stability[J].Food Chemistry,2016,192:178-187.DOI:10.1016/j.foodchem.2015.06.109.
[25]YANG Zhenzhong,WANG Yingchao,WENG Yi,et al.Bioassayguided screening and isolation ofα-glucosidase and tyrosinase inhibitors from leaves of Morus alba[J].Food Chemistry,2012,131(2):617-625.
[26]姚惠芳,景浩.笃斯越橘花青素与牛血清白蛋白的相互作用[J].食品科学,2013,34(23):6-10.DOI:10.1016/j.foodchem.2011.09.040.
[27]JESZKA-SKOWRON M,FLACZYK E,JESZKA J,et al.Mulberry leaf extract intake reduces hyperglycaemia in streptozotocin(STZ)-induced diabetic rats fed high-fat diet[J].Journal of Functional Foods,2014,8:9-17.DOI:10.1016/j.jff.2014.02.018.
[28]刘硕,王萌,朱少华,等.紫甘薯花色素与胰蛋白酶相互作用特性[J].食品科学,2014,35(23):232-237.DOI:10.7506/spkx1002-6630-201423045.