基于多光谱技术对原儿茶酸抑制低密度脂蛋白氧化及其与牛血清白蛋白相互作用的研究

The Inhibition Effect of Protocatechuic Acid on Low-Density Lipoprotein Oxidation and the Interaction Between This Acid With Bovine Serum Albumin Based on Multi-Spectral Techniques

植物多酚被称作人类健康的“第7类营养素”,在医药、食品和营养保健等多个领域受到广泛关注。红心猕猴桃果皮(RKP)中多酚含量丰富,是提取植物多酚的优良原料,但常被视为加工废料遭弃用。该研究拟通过脉冲超声(PU)辅助天然深共晶溶剂(NADES)提取RKP多酚,并通过荧光光谱和紫外-可见光谱探究RKP中重点多酚—原儿茶酸(PCA)抑制低密度脂蛋白(LDL)氧化功效及其与牛血清白蛋白(BSA)的相互作用机制。结果表明,NADES对RKP多酚的提取率显著高于常规溶剂(如水或乙醇)。在超声功率400 W、料液比1﹕40(g·mL^(-1))、温度70℃、提取20 min及含水率20%(ω/ω)条件下,所筛选的6种NADES溶剂中,氯化胆碱-乙二醇组合提取率最高(29.84 mg GAE/g DW)。光谱实验结果表明,PCA具有较强的DPPH自由基清除能力,清除率最高可达94.39%。PCA能够显著延长LDL氧化过程中共轭二烯(CD)生成的延滞时间及达到峰值时间,有效抑制脂质氧化过程中脂褐素及总荧光产物生成,减少LDL氧化过程中色氨酸(Trp)及赖氨酸(Lys)残基的氧化修饰,显示出对LDL氧化极强的抑制效果。多光谱法对PCA与BSA相互作用研究发现,两者之间存在相互作用,具有较强的亲和力,且只有一个结合位点,疏水作用力在相互作用过程中起主要作用;相互作用后酪氨酸(Tyr)周围微环境几乎不发生改变,而Trp周围微环境极性降低、疏水性增加;三维荧光光谱进一步证明二者之间存在相互作用,并由此引起蛋白质结构发生变化;综合荧光光谱和紫外-可见光谱的实验结果,PCA与BSA相互作用猝灭机理为静态猝灭。该研究为开发RKP及PCA提供参考。

Plant polyphenols,known as“Category 7th nutrients”for human health,have received widespread attention in many fields,including medicine,food and nutrition.However,red-fleshed kiwifruit peel(RKP),which contains many polyphenols and is known as an excellent raw material for extracting plant polyphenols,is often discarded as a by-product.First,the polyphenols were extracted by pulsed ultrasound(PU)assisted with natural deep eutectic solvent(NADES)from RKP.Subsequently,protocatechuic acid(PCA),the critical polyphenolin RKP,was selected to investigate the inhibition effect on low-density lipoprotein(LDL)oxidation and to investigate the interaction mechanism with bovine serum albumin(BSA)by fluorescence spectroscopy and UV-Vis spectroscopy.The results were as follows:The extraction rate of polyphenols by NADES was significantly higher than that of conventional solvents.Among the 6 kinds of NADES solvents screened,the highest extraction rate of polyphenols from RKP(29.84 mg GAE/g DW)was obtained with choline chloride-ethylene glycol under the conditions of ultrasonic power of 400 W,material-to-liquid ratio of 1∶40(g·mL^(-1)),temperature of 70℃,extraction time of 20 min and water content of 20%(ω/ω).PCA had a strong scavenging ability on DPPH radicals,with the highest scavenging rate of 94.39%in the results of the spectroscopic experiments.PCA could significantly prolong the delay time of conjugated diene(CD)production and peak value during LDL oxidation,effectively inhibit the production of lipofuscin and total fluorescent products during lipid oxidation and reduce the oxidation of tryptophan(Trp)residues and oxidative modification of lysine(Lys)residues during LDL oxidation.These spectroscopic experiments showed a strong inhibition effect on LDL oxidation.The interaction between PCA and BSA was studied,and the following results were obtained using multispectral techniques.The interaction and the strong affinity between PCA and BSA occurred;there was only one binding site,and the hydrophobic force played a major role in the interaction process.The microenvironment around Tyrosine(Tyr)was almost unchanged after the interaction,while a reduction of polarity and enhanced hydrophobicity around Trp residues occurred.The interaction between these two molecules was further verified in the three-dimensional fluorescence spectra,and the protein structure of BSA was changed after this interaction occurred.The quenching mechanism of interaction between PCA and BSA was static quenching according to these fluorescence and UV-Vis spectra experiments.This study provides new insights for the development of RKP and PCA.