【作者】 李莹；

【导师】 赵谋明；

【作者基本信息】 华南理工大学 ， 食品科学， 2011， 博士

【摘要】 本文以发酵酱油的生理活性物质为研究对象,在综合评价酱油基本成分指标及抗氧化活性的基础上,利用有机溶剂分级、正相硅胶柱层析、薄层制备层析、核磁共振、高分辨质谱、单晶X射线衍射、二极管阵列液质色谱、气相色谱-质谱联用等分离和鉴定手段,结合细胞模型评价体系,从发酵酱油中分离鉴定得到一种抗溃疡成分和四种具有诱导Ⅱ相酶活性的成分,并探讨其中的β-咔啉在食品模型体系中的形成规律及影响因素,最后对活性成分的分子作用机制进行深入研究,得出Ⅱ相酶诱导剂通过代谢激活和细胞信号传导的途径提高Ⅱ相酶表达的模型。采用主成分分析(PCA)、聚类分析(CA)和偏最小二乘回归(PLS)等多元回归分析法,探索了41种市售酱油样品的基本成分与抗氧化性的内在关系。结果表明,生产地区、发酵工艺和酱油类型对酱油的品质有显著影响;CA综合各指标对样品归类,评价出抗氧化能力优异的样品集群;根据PLS的分析,对抗氧化性起主要贡献的两个指标是酱油固形物和褐变指数。运用现代分离鉴定技术对华南地区生产的高盐稀态酱油进行分离纯化,首次在酱油乙酸乙酯提取物(EAE)的非极性部分中鉴定出一种抗溃疡成分——双环内酯素(dihydrocanadensolide),建立了以高精仪器为基础的相关鉴别和科学定量方法。双环内酯素在酱油发酵期间不断累积,发酵90天其在酱醪中的浓度可达169mg/L。在6个月的酱油储存期中,双环内酯素的稳定性较好,其含量在在冷藏储存(4℃左右)的样品中无显著变化,而在室温储存的样品中仅下降了14%。采用Hepa 1c1c7细胞醌还原酶(QR)活性示踪法,结合柱色谱、薄层制备色谱、核磁共振、高分辨质谱等分离和鉴定技术,从酱油EAE的弱极性部分中分离得到4种QR诱导成分:儿茶酚、大豆苷元、flazin和perlolyrin(后两者为β-咔啉类化合物),它们使细胞的QR活性加倍所需的浓度(CD值)依次为8μM、35μM、42μM和2μM。蛋白免疫印迹证实,这些活性物质诱导QR酶的表达量与诱导QR酶活性相一致。其中,β-咔啉类化合物的QR诱导活性为首次报道。研究了水相体系条件对四氢β-咔啉产物的形成和QR诱导活性的影响。结果表明,低pH与高温更利于Pictet-Spengler反应的进行,色氨酸与羟甲基糠醛在体系pH是1、4、7时所得产物的CD值分别为4.0、25.1、46.7μg/mL,而pH值为10时,反应产物不存在CD值。色氨酸与香兰素的反应产物也有较好的QR诱导活性(CD值为11.8μg/mL)。此外,基于Pictet-Spengler原理对perlolyin进行有机合成,所得的合成化合物与天然分离化合物的QR诱导活性一致。以Hepa 1c1c7和Hep G2细胞模型,探讨了酱油癌症预防成分的分子作用机理。结果表明,儿茶酚的作用机制中包含非典型的氧化应激过程,而Flazin、perlolyrin和大豆苷元则产生氧化还原信号。Flazin和perlolyrin的活性发挥须经细胞色素P450的1A2亚族酶进行代谢激活。Perlolyrin的信号传导经由PI3K通路,但未影响转录因子Nrf2的核转录水平;儿茶酚的信号传导是通过抑制p38激酶信号通路,上调了Nrf2的核转移量从而增加了QR酶的表达;flazin则是激活了p38信号通路增强了QR酶的表达,且Nrf2也是其信号转递的一个重要环节。更多还原

【Abstract】 The present study focused on isolation, identification and mechanistic actions of bioactive compounds in soy sauce. Firstly, multivariate statistical analysis was performed on commercial soy sauce samples to find that traditional fermented soy sauce is the most appropriate subject for further study. Secondly, bioactive compounds with activities of anti-ulcer and quinone reductae (QR) induction were isolated and identified from nonpolar and semipolar fractions of fermented soy sauce. Thirdly, the formation conditions ofβ-carbolines, the newly found QR inducers, were studied in a food model system. Lastly, molecular mechanism of QR inducers was investigated.Antioxidant activities (AA) and quality indices were determined in forty-one commercial soy sauces. The relationships between quality indices and AA were elucidated by multivariate analysis techniques. Results showed that production location, fermentation process and styles had significant (P<0.05) impacts on the characteristics of soy sauces. Satisfactory discrimination between dark and light soy sauces was achieved by principal component analysis. Using cluster analysis, the group of dark soy sauce with strong AA was obtained. Results from partial least squares regression (PLS) showed that TS and BI were two major indices contributing to the AA of soy sauce.Ethyl acetate extract of HSHW fermented soy sauce was separated by column chromatography. Collective techniques of nuclear magnetic resonance (NMR), mass spectrometry (MS) and single-crystal X-ray diffraction were used to identify dihydrocanandensolide, an anti-ulcer agent, from non-polar column fractions Moreover, quantitative study showed that concentration of dihydrocanadensolide increases the fermentation period of soy sauce. After 90 days, the maximum concentration of dihydrocanadensolide in moromi of soy sauce reached 169 mg/L. In addition, stability of dihydrocanadensolide in soy sauce samples stored at 4°C was better than those stored at room temperature.A murine hepatoma (Hepa 1c1c7) cellular bioassay was used to guide the isolation of phase II enzyme inducers from fermented soy sauce, using quinone reductase (QR) as a biomarker. Four most potent compounds, catechol, daidzein, flazin and perlolyrin, were identified with respective CD values (concentration required to double QR) of 8μM, 35μM, 42μM and 2μM. Western blots confirmed that the enhancement of QR activities of the four QR inducers corresponded to a dose-dependent increases in cellular levels of NAD[P]H:quinone Oxidoreductase 1 protein.Perlolyrin was organically synthesized according to Pictet-Spengler reaction. Inspired by this process, formation of tetrahydro-β-carboline was studied in an aqueous system. Low pH and high temperature conferred the reactions between tryptophan (Trp) and hydroxymethylfurfural (HMF). In consistent with this result, QR inducing activity of products decreased while increasing pH in the reacting system. Furthermore, no CD value was observed under basal conditions (pH 10). Interestingly, reacting product of Trp and vanillin at pH1 also exhibited very potent QR inducing activity with a CD value 11.8μg/mL.Results from co-addition of glutathione, N-acetyl cysteine and ascorbic acid indicated that oxidative stress was involved in the induction of QR by catechol, while redox signals activated QR inductions of flazin, perlolyrin and daidzein. Cytochrome P450 (CYP) isomer 1A2 is required for QR induction of both flazin and perlolyrin. Studies with signaling pathway inhibitors and western blots suggested that chemical signal of catechol was transducted through inhibiting p38 kinase pathways, and redox signal of flazin passed through p38 pathways, while perlolyrin induce QR by activation of PI3K pathway. Role of transcription factor NF-E2-related factor 2 (Nrf2) is critical in QR induction by catechol and flazin.To our knowledge, this is the first report on the presence of dihydrocanadensolide in food system, the ability ofβ-carboline-derived alkaloids to induce phase II enzymes and mechanistic studies on their QR induction activities.更多还原