【作者】 王强；

【导师】 陈秀敏；

【作者基本信息】 江苏大学 ， 食品科学与工程， 2022， 硕士

【摘要】 纳米氧化锌(ZnO NPs)具有抗癌和药物递送载体的重要功能。传统的物理和化学合成法存在污染大、耗能高和威胁人体健康等问题,而利用植物提取物绿色合成ZnO NPs具有高效、环保、无毒和成本低等优点。超声辅助合成ZnO NPs能够加快反应速率、减小粒径和提高产率。咖啡叶作为一种咖啡树的低值副产物含有丰富的多酚类化合物、糖和蛋白质等物质,因此能够为绿色合成ZnO NPs提供大量的还原剂、稳定剂、螯合剂和封盖剂。芒果苷(MGF)是一种具有抗癌和抗炎等活性的多酚类物质,但溶解度和生物利用度低,从而影响其生物活性。本论文研究超声辅助咖啡叶提取物绿色合成ZnO NPs的条件并解析合成的ZnO NPs的特征,然后用合成的ZnO NPs负载芒果苷并研究其对癌症细胞Hep G2和Caco-2的抑制作用。本文主要的研究结果如下:(1)通过单因素实验筛选出ZnO NPs产率最高的合成条件为:p H值8,咖啡叶与乙酸锌质量比1.71:1,超声时间10 min,超声频率28/40 k Hz,超声功率180W,合成温度30℃。此条件下ZnO NPs的产率(43.59%)显著高于对照组未经超声合成(41.45%)和化学方法合成的产率(24.79%)。因此,超声辅助咖啡叶提取物绿色合成法不仅可以缩短合成时间和降低合成温度,而且还能够提高ZnO NPs的合成产率。(2)通过比较超声和未经超声对照组两种反应体系中总酚含量(TPC),发现TPC在超声合成体系中降低了98.16%,其下降程度显著高于未经超声体系的97.10%。进一步分析植化成分发现多酚类化合物包括芒果苷、芦丁和绿原酸在这两个反应体系中降低了95.29%-100%,而葫芦巴碱和咖啡因的含量仅下降了18.47%-36.06%,表明多酚类化合物特别是5-咖啡酰奎宁酸(5-CQA)是合成ZnO NPs的主要还原剂和螯合剂。研究还发现5-CQA、葫芦巴碱和咖啡因在超声合成体系中的含量显著(P<0.05)低于未经超声体系。通过UV-Vis、SEM、EDX、TEM、FTIR、XRD和Zeta电位表征发现超声辅助合成的ZnO NPs的平均粒径为8.29nm,显著小于未超声合成(10.48 nm)和化学合成的粒径(17.15 nm)。三种方法合成的ZnO NPs均呈类似球形,均为六方晶系纤锌矿结构,结晶性好,纯度高。(3)MGF-ZnO NPs的载药量和包封率分别为3.66%和19.01%,132 h后的体外累积释放率为96.24%。MGF-ZnO NPs能显著降低Hep G2和Caco-2细胞的存活率;降低细胞内的p H值;增加细胞膜通透性从而提高乳酸脱氢酶(LDH)的释放率;增加细胞内的活性氧自由基(ROS)水平进而诱导细胞产生氧化应激;促进细胞染色质浓缩和细胞核裂解;提高细胞的总体凋亡比例以及使Hep G2和Caco-2细胞分别发生G2/M期和S期细胞周期阻滞。因此,MGF-ZnO NPs可增强对癌细胞的抑制作用,具有抗癌的应用前景。更多还原

【Abstract】 Zinc oxide nanoparticles(ZnO NPs)are function as usually anticancer drugs and drug delivery carriers.The traditional physical and chemical methods used to synthesize ZnO NPs have several drawbacks such as serious pollution,high energy consumption,and harm for human health;however the green synthesis of ZnO NPs using plant extract has the advantages of high efficiency,environmental friendly,nontoxicity,and low cost.Ultrasound-assisted synthesis of ZnO NPs can accelerate the reaction rate,reduce particle size,and improve yield.As a low-value by-product of coffee tree,coffee leaves are rich in polyphenolic compounds,sugars,and proteins,which can provide a large amount of reductants,stabilizing agents,chelating agents,and capping agents for green synthesis of ZnO NPs.Mangiferin(MGF)is a polyphenolic substance with anticancer and anti-inflammatory activities;however,its low solubility and bioavailability affect its biological activity.In this paper,we investigated the conditions of ultrasound-assisted green synthesis of ZnO NPs using coffee leaf extract and analyzed the characteristics of as-synthesized ZnO NPs.Thereafter,mangiferin was loaded on the as-synthesized ZnO NPs and the inhibitory effects on cancer cells,including Hep G2 and Caco-2 were investigated.The main findings of this paper are as follows:(1)One factor at a time experimental design was used to screen the synthesis conditions with the highest yield of ZnO NPs,which include: p H value,8;mass ratio of coffee leaf to zinc acetate,1.71 : 1;ultrasound time,10 min;ultrasound frequency,28/40 k Hz;ultrasound power,180 W;and synthesis temperature,30 ℃.The yield of ZnO NPs synthesized under this condition(43.59%)was significantly higher than that of the control group non-ultrasound-assisted synthesis(41.45%)and chemical synthesis methods(24.79%).Therefore,ultrasound-assisted green synthesis of ZnO NPs using coffee leaf extract can not only shorten the synthesis time and reduce the synthesis temperature,but also improve the synthesis yield.(2)By comparing the total phenolic content(TPC)in the two reaction systems with and without ultrasound treatment,it was found that TPC decreased by 98.16% in the ultrasound-assisted synthesis system,and the degree of decrease was significantly higher than that in the non-ultrasound-assisted synthesis system(97.10%).Further analysis of phytochemical components found that polyphenolic compounds including mangiferin,rutin,and chlorogenic acid were reduced by 95.29%-100% in these two reaction systems,while the content of trigonelline and caffeine was only reduced by18.47%-36.06%,indicating that polyphenols,especially 5-caffeoylquinic acid(5-CQA),were the main reductants and chelating agents for the synthesis of ZnO NPs.The study also found that the contents of 5-CQA,trigonelline,and caffeine in the ultrasoundassisted synthesis system were significantly(P < 0.05)lower than that in the nonultrasound-assisted synthesis system.After characterization of ZnO NPs synthesized in these three reaction systems using UV-Vis,SEM,EDX,TEM,FTIR,XRD,and Zeta potential,it was found that the average particle size of the ultrasound-assisted synthesized ZnO NPs was 8.29 nm,which was significantly smaller than ZnO NPs synthesized by the non-ultrasound-assisted(10.48 nm)and chemical methods(17.15nm).The ZnO NPs synthesized by the three methods were all spherical-like in shape,with hexagonal wurtzite structure,good crystallinity,and high purity.(3)The drug loading and encapsulation efficiency of MGF-ZnO NPs were 3.66%and 19.01%,respectively,and the in vitro cumulative release of MGF after 132 h was96.24%.MGF-ZnO NPs significantly reduced the viability of Hep G2 and Caco-2 cells;decreased the intracellular p H;increased the cell membrane permeability,thereby increasing the release of lactate dehydrogenase(LDH);increased the intracellular reactive oxygen species(ROS)level and then leading to the oxidative stress in cells;promoted cellular chromatin condensation and nuclear lysis;increased the overall apoptotic ratio of cells and induced cell cycle arrest in G2/M phase and S phase in Hep G2 and Caco-2 cells,respectively.Therefore,MGF-ZnO NPs can enhance the inhibitory effect on cancer cells,therefore,they are considered promising anticancer drugs.更多还原