The mechanism of interactions between tea polyphenols and porcine pancreatic alpha‐amylase: Analysis by inhibition kinetics,fluorescence quenching,differential scanning calorimetry and isothermal titration calorimetry

1 Scope

This study aims to use a combination of biochemical and biophysical methods to derive greater mechanistic understanding of the interactions between tea polyphenols and porcine pancreatic α‐amylase (PPA).

2 Methods and results

The interaction mechanism was studied through fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) and compared with inhibition kinetics. The results showed that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red‐shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also suggested by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between the reciprocal of competitive inhibition constant (1/K_ic), quenching constant (K_FQ) and binding constant (K_itc).

3 Conclusion

The combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA.     

Conformation change of trypsin induced by acteoside as studied using multiple spectroscopic and molecular docking methods

The interaction of trypsin with acteoside was studied using ultraviolet visible absorption, fluorescence, synchronous fluorescence, circular dichroism techniques, along with molecular docking method. The fluorescence experiments indicated that acteoside quenched the intrinsic fluorescence of trypsin via a combined quenching process (static and dynamic quenching). The binding constant of acteoside to trypsin obtained was 2.50 × 10⁵ L mol^?1 at 298 K and the number of binding site was about one under the same experimental condition. The thermodynamic functions ΔH° and ΔS° of the binding process were 8.79 kJ mol^?1 and 132.58 J mol^?1 K^?1, respectively, which indicated that the hydrophobic force was the main acting force between them. Ultraviolet–visible, synchronous fluorescence together with circular dichroism spectra studies demonstrated that the interaction of acteoside with trypsin lead to a loosening and unfolding of the protein backbone with partial β-sheet structures being transformed into α-helix structures. All these experimental results were validated and explained reasonably by docking studies. And the molecular docking results further illustrated that besides hydrophobic forces, hydrogen bonds also played an important role in the stabilization of the acteoside–trypsin complex. Results from this study should be helpful to make full use of acteoside in the food industry and be useful to the design of the drugs for the diseases related to trypsin.     

Regulating inhibitory activity of potato I-type proteinase inhibitor from buckwheat by rutin and quercetin

This study aims to investigate the effects of two flavonoids, rutin and quercetin, on inhibitory activity of recombinant buckwheat trypsin inhibitor (rBTI). We found that rutin and quercetin could quench the florescence of rBTI through the static quenching process. We also observed that upon binding to rutin or quercetin, rBTI underwent conformational changes. The results also suggested that rutin and quercetin bind to two different sites on rBTI through different interactions: rutin binds to rBTI through van der Waals forces and hydrogen bonds, whereas quercetin binds through hydrophobic interactions. Rutin and quercetin also markedly deactivated the trypsin inhibitory activity (TIA) of rBTI, while quercetin exhibited higher inactivation effect on rBTI than rutin due to its structure. Finally, the molecular docking revealed the molecular binding between the flavonoids and rBTI. These findings can be useful for the understanding of how flavonoid affects the inhibitory of rBTI.     

柚皮素对α-葡萄糖苷酶的抑制作用及其机制

为研究柚皮素对α-葡萄糖苷酶的抑制作用,本文采用酶动力学、荧光光谱和分子对接等方法研究了柚皮素对α-葡萄糖苷酶的抑制效果、抑制作用类型及其抑制作用的分子机制。柚皮素对α-葡萄糖苷酶的IC₅₀为0.174 mmol/L,显著低于阿卡波糖的0.721 mmol/L,为非竞争型抑制剂,K_i值为0.114 mmol/L;柚皮素和α-葡萄糖苷酶的结合导致了酶分子的内在荧光静态猝灭,猝灭常数为0.1598×104 L/mol,结合位点数n为1。分子对接结果显示,在氢键、离子键、疏水作用、π-π T型堆积、静电作用五种作用力的驱动下,柚皮素结合于α-葡萄糖苷酶分子的一个疏水口袋中,结合能为?7.6 kJ/mol。本文研究结果表明,柚皮素是一种较好的食源性α-葡萄糖苷酶抑制剂,在辅助治疗糖尿病功能食品中具有良好的应用前景。     

紫甘薯花色素与胰蛋白酶相互作用特性

测定紫甘薯花色素与胰蛋白酶反应前后酶的催化活性、催化反应动力学并采用紫外光谱法、荧光光谱法和红外光谱法研究紫甘薯花色素与胰蛋白酶相互作用特性。结果表明：紫甘薯花色素对胰蛋白酶催化活性有明显的抑制作用,抑制类型为可逆的竞争性抑制,抑制常数Ki＝6.16×10－4 mmol/L,当紫甘薯花色素与胰蛋白酶的物质的量比为140∶1,在 37 ℃反应 15 min,抑制率达到38.61%,而反应时间对催化活性的影响不明显;紫甘薯花色素可使胰蛋白酶的内源荧光猝灭,猝灭类型为静态猝灭,室温下猝灭常数Kq为1.73×1012 L/（mol·s）,结合常数KA为3.88×104 L/mol,结合位点数n为0.86;热力学参数确定两者之间的作用力主要为氢键和范德华力;根据Förster能量转移理论得出它们的结合距离为3.56 nm;红外光谱经过去卷积、二阶导数处理得知与紫甘薯花色素作用后胰蛋白酶的α-螺旋含量降低,β-折叠含量升高。     

Investigation of the interaction between (−)‐epigallocatechin‐3‐gallate with trypsin and α‐chymotrypsin

Tea polyphenol (TP) inhibits digestive enzymes and reduces food digestibility. To explore the interaction between TP with digestive enzymes, bindings of ‐epigallocatechin‐3‐gallate (EGCG) to trypsin and α‐chymotrypsin were studied in detail using fluorescence, resonance light‐scattering, circular dichroism, fourier transform infrared spectroscopy methods and protein‐ligand docking. The binding parameters were calculated according to Stern–Volmer equation, and the thermodynamic parameters were determined by the van't Hoff equation. The results indicated that EGCG was capable of binding trypsin and α‐chymotrypsin with high affinity, resulting in a change of native conformation of these enzymes. EGCG had a greater influence on the structure of α‐chymotrypsin than trypsin. This study can be used to explain the binding interaction mechanism between TP and digestive enzymes.     

基于光谱和分子模拟研究半乳甘露聚糖和人血清白蛋白的相互作用

目的:研究半乳甘露聚糖对人血清白蛋白（HSA）光谱特性的影响及它们相互作用的机理。方法:本文利用光谱法判断半乳甘露聚糖和HSA的猝灭方式、结合位点数、结合作用力类型以及二级结构的变化,采用分子对接模拟技术得到结合作用力类型和长度,进一步研究半乳甘露聚糖和HSA相互作用的机制。结果:在半乳甘露聚糖的作用下,HSA内源荧光被有规律的猝灭,猝灭过程是自发进行的,机制为静态猝灭,结合位点数约为1,并且HSA的二级结构中α-螺旋含量减少了7.7%。分子对接结果表明,半乳甘露聚糖通过氢键和范德华力在HSA的亚结构域IIB中相互作用。结论:半乳甘露聚糖与HSA有较强的结合能力,并且结合是自发进行的。     

黄嘌呤氧化酶多酚抑制剂的筛选及其作用机制

黄嘌呤氧化酶(xanthine oxidase,XOD)是人体内次黄嘌呤和黄嘌呤代谢产生尿酸的关键酶,而多酚单体可抑制黄嘌呤氧化酶从而减少尿酸合成进而缓解痛风症状。为探究来源于天然产物的多酚单体对XOD的抑制作用机制,首先研究几种常见活性较好的多酚单体对黄嘌呤氧化酶的抑制率,及其对XOD的抑制动力学,并且通过荧光猝灭实验与分子对接模拟多酚单体与XOD之间的相互作用形式。在6种多酚单体中,白藜芦醇、表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)和橙皮苷对黄嘌呤氧化酶活性的抑制效果较好,室温下IC50分别为261.22、150.51、97.08μmol/L,是黄嘌呤氧化酶的竞争性抑制剂。荧光猝灭实验表明,单体与XOD结合使酶的内源荧光发生了猝灭现象,猝灭机制为静态猝灭。分子对接模拟表明,多酚单体能与XOD的氨基酸残基结合,结合位点位于钼蝶呤(molybdopterin,Mo-Pt)辅因子或黄素腺嘌呤二核苷酸(flavine-adenine dinucleotide,FAD)结构域的异恶嗪环附近。多酚单体是有效的XOD竞争性抑制剂,可以通过抑制黄嘌呤氧化酶的活性有效降低尿酸水平,在天然产物的开发和功能性食品的应用中具有很好的前景。     

槲皮素对酪氨酸酶的抑制作用与分子机理

本文以L-多巴为底物,采用酶抑制动力学法研究了槲皮素对酪氨酸酶的抑制作用大小及类型,并采用荧光光谱技术分析其与酪氨酸酶的猝灭作用类型、结合位点、作用力类型。在此基础上,进一步利用柔性分子对接技术分析槲皮素对酪氨酸酶的抑制机理。结果表明,槲皮素对酪氨酸酶具有抑制作用,抑制常数KI为36 m M,以竞争性抑制剂形式抑制酪氨酸酶活性,是一种可逆性抑制剂;槲皮素以1:1比例通过氢键和疏水作用力结合于酪氨酸酶活性中心,且对酪氨酸酶的荧光产生静态淬灭作用,具有氢键及疏水作用力;分子对接结果验证了以上实验结论:槲皮素占据了酪氨酸酶活性中心,且与活性中心部位的Asn260和Gly62残基形成了强烈的氢键作用,同时伴有疏水作用共同稳定复合物的结构。     

The interaction of sesamol with DNA and cytotoxicity,apoptosis, and localization in HepG2 cells

Sesamol, a nutritional antioxidant phenolic compound present in sesame seed, has a potential therapeutic molecule effect against cancers. In this study, the interaction between sesamol and DNA was investigated by employing ultraviolet/visible (UV/Vis), fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), and molecular modeling. The fluorescence analysis indicated that the fluorescence quenching mechanism of sesamol by calf thymus DNA (ctDNA) occurred through static quenching. The UV/Vis, CD, FT-IR spectra and molecular docking results implied that the primary binding mode was minor groove binding. Furthermore, the intracellular interaction of sesamol with DNA and its bioactivity effect were explored. The cell activity results demonstrated that sesamol induced hepatic cell line (HepG2) death. The acridine orange (AO)/ethidium bromide (EB) staining assay and DNA fragmentation confirmed that sesamol could efficiently induce the apoptosis of HepG2 cells. Moreover, addition of sesamol to HepG2 cells resulted in nuclear localization, as visualized by confocal microscopy.     

分子对接和荧光光谱法研究麦角甾醇与牛血清白蛋白的相互作用

用分子荧光光谱实验法和分子对接理论研究麦角甾醇与牛血清白蛋白（bovine serum albumin,BSA）的相互作用。荧光光谱实验结果表明：麦角甾醇能猝灭BSA的内源性荧光,其猝灭类型为静态猝灭;通过考察猝灭过程中热力学函数的变化初步推断麦角甾醇与BSA的结合是自发的熵增过程,驱动力主要为疏水相互作用。运用分子对接技术研究了麦角甾醇与BSA的相互作用,结果表明：麦角甾醇与BSA相结合,主要的作用力类型为疏水相互作用;并获得了麦角甾醇在BSA中的作用位点,麦角甾醇处在一个疏水性的结合口袋中,结合稳定性强。荧光光谱的实验结果与分子对接的理论结果总体上一致,说明结合过程是一个自发的过程,BSA可以携带和运输麦角甾醇,同时从分子对接中获得了麦角甾醇在BSA中详细的结合位点和结合模式。     

Study on the interaction of food colourant quinoline yellow with bovine serum albumin by spectroscopic techniques

The interaction of a food colourant, quinoline yellow (Qy), and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorometry, FT-IR and circular dichroism (CD) techniques. The experimental results indicated that the quenching mechanism of BSA by the dye was a static procedure. Various binding parameters were evaluated. The negative value of ΔH, negative value of ΔS and the negative value of ΔG indicated that van der Waals force and hydrogen bonding play major roles in the binding of Qy and BSA. Based on Forster's theory of non-radiation energy transfer, the binding distance, r, between the donor (BSA) and acceptor (Qy) was evaluated. The results of CD and UV-vis spectroscopy showed that this dye could bind to BSA and the conformation of BSA changed.     

仿刺参胶原蛋白源二肽基肽酶抑制肽虚拟筛选及分子对接研究

目的 采用生物信息学和分子对接方法筛选仿刺参胶原蛋白来源的二肽基肽酶(dipeptidyl peptidase-IV, DPP-IV)抑制肽。方法 以仿刺参胶原蛋白序列为对象,进行生物信息学分析和计算机辅助虚拟酶解, 基于生物毒性、致敏性、水溶性及吸收、分配、代谢、排泄及毒性(absorption, distribution, metabolism, excretion, toxicity, ADMET)预测, 筛选得到6条具有潜在生物活性的寡肽, 氨基酸序列分别为CD、CQ、CS、GR、SM、MDG, 进一步通过分子对接分析肽段与DPP-Ⅳ的结合活性, 并分析其结合位点及方式。结果 生物信息学分析表明仿刺参胶原蛋白是生物活性肽的潜在优良来源, 经木瓜蛋白酶、碱性蛋白酶及模拟胃肠道虚拟水解后能够释放出DPP-Ⅳ抑制肽; 分子对接表明俩条新肽段CS、SM通过氢键、疏水相互作用分别与DPP-IV的S1、S2活性口袋结合。结论 本研究提供了一种快速筛选刺参胶原蛋白中DPP-IV抑制肽的方法。     

黄曲霉毒素G_1与人血清白蛋白的结合机理及分子对接

在模拟人体血液p H 7.4的条件下,用分子对接及其荧光光谱、3D荧光、圆二色谱等方法研究黄曲霉毒素G1(aflatoxin G_1,AFG_1)与人血清白蛋白(human serum albumin,HSA)的相互作用。结果发现,根据双对数方程得出AFG1与HSA结合反应猝灭机制为静态猝灭,4个温度条件下结合常数数量级均为104,结合位点数近似为1。通过分子对接和热力学参数计算,AFG1结合在HSA的ⅠB疏水腔中,二者结合力主要为疏水作用和氢键。通过研究体内金属离子对AFG1-HSA反应的影响,Fe~(3+)、Mg~(~(2+))能增大AFG1对HSA的亲和力,而Zn~(2+)、Cu~(2+)、Mn~(2+)离子则能大大降低AFG1与HSA的亲和力。基于F?rster’s能量转移,二者反应距离为3.26 nm。3D荧光结果显示,二者的结合反应使HSA生色团氨基酸残基疏水性增加,二级结构发生改变;圆二色谱结果表明,加入AFG1使得HSA的α-螺旋含量增加。     

芹菜素-8-C-葡萄糖苷对淀粉消化酶抑制作用机制

采用酶活动力学、荧光光谱、圆二色谱和分子对接等技术系统探究芹菜素-8-C-葡萄糖苷对α-淀粉酶和α-葡萄糖苷酶活性调控效果及机制。结果显示,芹菜素-8-C-葡萄糖苷对α-葡萄糖苷酶有良好的抑制效果,IC50值为293.5 mg/L,抑制类型为非竞争性抑制。但对α-淀粉酶无显著抑制效果。荧光光谱结果表明芹菜素-8-C-葡萄糖苷可作为猝灭剂分子与α-葡萄糖苷酶结合,发生静态猝灭,改变酶蛋白氨基酸疏水环境。圆二色谱则显示芹菜素-8-C-葡萄糖苷和α-葡萄糖苷酶之间的相互作用使酶分子的二级结构变得松散,α-螺旋和β-转角下降。分子对接结果进一步证实芹菜素-8-C-葡萄糖苷和α-葡萄糖苷酶之间作用力主要为氢键,最低结合能为-7.2 kcal/mol。本研究揭示了芹菜素-8-C-葡萄糖苷对淀粉消化酶尤其是α-葡萄糖苷酶的抑制作用机制,为未来将芹菜素-8-C-葡萄糖苷作为健康食品辅料或药物开发提供一定理论基础。     

Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods

The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV–vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA.     

异甘草素抑制α-葡萄糖苷酶的分子机制

α-葡萄糖苷酶活性与糖尿病患者的餐后血糖水平有重要关联,寻找食源性的α-葡萄糖苷酶抑制剂是当前功能性食品研究的热点。异甘草素是甘草的重要活性成分,相关研究表明甘草提取物具有α-葡萄糖苷酶抑制活性,推测与异甘草素有关。鉴于此,本实验通过酶抑制、荧光猝灭以及分子对接等方法研究异甘草素抑制α-葡萄糖苷酶活性的机制。结果表明,异甘草素以竞争性与非竞争性相混合的方式抑制α-葡萄糖苷酶,其抑制效果明显优于阿卡波糖。荧光猝灭分析结果表明在疏水作用力驱动下异甘草素可与α-葡萄糖苷酶结合生成复合物,结合位点数为1。分子对接结果验证了相关实验结论：异甘草素位于酶的疏水口袋中,与残基Asp202和Arg400以氢键结合,并与周围众多的疏水残基存在疏水作用,共同维持该复合物结构。本研究对于开发新型的食源性α-葡萄糖苷酶抑制剂、推动异甘草素在功能性食品和医药领域的应用具有一定的参考意义。     

蒲公英水提多酚的脲酶抑制作用及机理

蒲公英水提多酚具有很高的抑制脲酶活性的潜力,本文以蒲公英全草粉为材料,采用液质联用法（High Performance Liquid Chromatography-Mass Spectrometry,HPLC-MS）、苯酚-次氯酸盐比色法、Lineweaver-Burk双倒数作图法、荧光分析法和分子对接筛选蒲公英中抑制脲酶活性的主要水提多酚,并分析其抑制效果和作用机制。结果表明,蒲公英水提多酚主要成分是菊苣酸和咖啡酸,含量分别为10.62%和7.39%,结合能量为?5.65和?3.60 kcal/mol,对脲酶的抑制率之和占蒲公英水提总多酚抑制率的75.76%,是抑制脲酶活性的主要成分;半数抑制浓度（the half maximum inhibitory concentration,IC₅₀）分别为0.34±0.07和3.04±0.68 mmol/L,抑制效果呈浓度依赖性和低浓度高效性;两者与脲酶的作用位点相同,抑制类型均为以氢键和范德华力为主的非竞争性抑制类型,且荧光猝灭机制为静态猝灭。本研究为阐明蒲公英中抑制脲酶的主要多酚及作用机理,促进多酚类脲酶抑制剂的开发提供了理论依据。     

Changes in inhibitory activity and secondary conformation of soybean trypsin inhibitors induced by tea polyphenol complexation

BACKGROUND: Tea polyphenol (TP) is a new food additive for antioxidant application, while soybean is an important resource for food and feed processing. It is therefore of rational and practical significance to investigate the influence of TP on soybean trypsin inhibitors (TIs). The aim of this study was to determine the effects of TP on the inhibitory activity of Kunitz (KTI) and Bowman–Birk (BBTI) TIs and to reveal the relationship between the inhibitory activity and conformation of KTI and BBTI by measurement of circular dichroism (CD) spectra. RESULTS: KTI and BBTI were found to be partially deactivated by TP. BBTI exhibited stronger resistance than KTI to TP deactivation. The unchanged K_M value of trypsin for benzoyl‐DL ‐arginine‐p‐nitroanilide hydrolysis indicated that KTI and BBTI inhibited trypsin in a non‐competitive pattern when complexed with TP. As the TP/TI ratio was increased and the inhibitory activity of KTI and BBTI decreased, the conformation of KTI and BBTI showed relevant changes and the major CD negative bands shifted progressively towards the near‐UV region. CONCLUSION: These results show the deactivation effects of TP on KTI and BBTI and reveal preliminarily the relationship between the inhibitory activity and secondary structure of KTI and BBTI. Copyright © 2009 Society of Chemical Industry     

豆粕血管紧张素转化酶抑制肽的结构鉴定及作用机制解析

以大豆粕为原材料,利用超声辅助酶解技术、超滤-?KTA层析相结合的方法分离纯化获取豆粕酶解产物中血管紧张素转化酶（angiotensin-converting enzyme,ACE）抑制肽,对其分子质量分布进行研究,后通过质谱分析与分子对接技术鉴定并筛选出ACE抑制活性肽的氨基酸序列,经固相合成肽序列,检测其ACE抑制肽的活性并基于分子对接技术探索其抑制机制。结果表明：经超声辅助酶解提取获得的豆粕肽分子质量主要分布在6 000 Da以下;根据分子对接的最低预测自由能筛选出的GVRP（－8.44 kcal/mol）和IIVTP（－9.04 kcal/mol）可以抑制ACE活性,半抑制浓度（50% inhibitory concentration,IC50）分别为（84±0.06）、（77±0.08）μmol/L;分子对接结果表明：GVRP、IIVTP能够与ACE的活性口袋S1、S1′、S2形成氢键相互作用,共有的过近接触（3.5 ?范围内）ACE氨基酸残基为His513、Ala354和Glu384。本研究基于串联质谱与分子对接技术,建立从混合多肽中快速鉴定、筛选活性多肽的方法,探究活性多肽与ACE稳定结合并体现其ACE活性的抑制机制,为后续的深入研究提供一定参考。