Isolation, Identification And Mechanistic Actions Of Bioactive Compounds In Traditional Fermented Soy Sauce

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.