肠道菌群酵解对红枣多酚及其保护氨基甲酸乙酯所致氧化损伤的影响

Phenolic profile of jujube fruit subjected to gut microbiota fermentation and its antioxidant potential against ethyl carbamate-induced oxidative damage 肠道菌群酵解对红枣多酚及其保护氨基甲酸乙酯所致氧化损伤的影响 1 first-author 1 1 1 2 corresp Wei CHEN, zjuchenwei@zju.edu.cn Wei CHEN, zjuchenwei@zju.edu.cn zjuchenwei@zju.edu.cn https://orcid.org/0000-0002-2373-2437 Department of Food Science and Nutrition, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou310058, China Department of Food Science and Nutrition, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University, Hangzhou310058, China Ningbo Research Institute, Zhejiang University, Ningbo315100, China Ningbo Research Institute, Zhejiang University, Ningbo315100, China Objective 2 To evaluate the composition of bioactive substances and the antioxidant effects of jujube fruit under gut microbiota fermentation (GMF), and the inhibitory effect on cytotoxicity caused by ethyl carbamate (EC). Methods 2 Changes in the contents of flavonoids, polyphenols, total sugars, and reducing sugars of jujube fruit after GMF (0, 2, 6, 12, 24, and 48 h) were determined. The oxidation resistance of fermented jujube fruits (from 0 to 48 h fermentation) was evaluated using in vitro 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) and ferric reducing antioxidant power (FRAP) assays. Inhibitory effects of 48 h-fermented jujube fruit at various concentrations (0.25, 0.50, 1.00, and 2.00 mg/mL) on EC-treated toxicity and DNA damage of Caco-2 cells were estimated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and nuclear staining assays, respectively. Effects of different concentrations of jujube fruit on EC-treated Caco-2 cells’ intracellular reactive oxygen species (ROS), glutathione (GSH) levels, and mitochondrial membrane potential (MMP) were also evaluated. Results 2 Jujube fruit has rich bioactive components after GMF and shows strong antioxidant capacity. Fermented jujube fruit can inhibit the cytotoxicity and DNA damage of Caco-2 cells caused by EC and reduce intracellular ROS generation, as well as restoring GSH and MMP. Conclusions 2 Fermented jujube fruit extracts produced by GMF still contain biologically active substances which retain biological activity and antioxidation capabilities. 目的 2 研究红枣在肠道菌群酵解作用下的生物活性物质组成和抗氧化功效，以及其对氨基甲酸乙酯诱导细胞毒性的保护作用，为红枣在肠道菌群酵解后发挥抗氧化作用提供新思路。 创新点 2 首次研究了肠道菌群酵解对红枣中生物活性物质组成和抗氧化能力的影响及其对氨基甲酸乙酯诱导细胞毒性的保护作用。 方法 2 红枣模拟肠道菌群酵解（0~48 h）后，采用比色法和高效液相色谱法分析黄酮、多酚、总糖和还原糖在发酵过程中的含量变化；采用体外抗氧化活性评价方法研究红枣发酵代谢产物抗氧化活性；采用MTT法和Hoechst 33258荧光探针研究不同浓度（0.25、0.50、1.00和2.00 mg/mL）红枣发酵48 h代谢产物对氨基甲酸乙酯诱导Caco-2细胞的细胞活力和DNA损伤的影响；分别采用6-羧基-2’,7’-二氯二氢荧光素二乙酸酯（DCFH-DA）和萘-2,3-二甲醛（NDA）荧光探针检测不同浓度（0.25、0.50和1.00 mg/mL）红枣发酵48 h代谢产物对氨基甲酸乙酯诱导Caco-2细胞内活性氧（ROS）和谷胱甘肽（GSH）水平；采用RH123荧光探针检测不同浓度（0.25、0.50和1.00 mg/mL）红枣发酵48h代谢产物对氨基甲酸乙酯诱导Caco-2细胞内线粒体膜电位的影响。 结论 2 红枣经肠道菌群酵解后含有丰富的生物活性成分，具有很强的抗氧化能力。红枣发酵代谢物可抑制氨基甲酸乙酯诱导的Caco-2细胞活力下降和DNA损伤，减少细胞内ROS生成，恢复GSH水平和线粒体膜电位，抑制氨基甲酸乙酯诱导的细胞氧化损伤。 Jujube Gut microbiota fermentation Polyphenols Ethyl carbamate Antioxidant activity 红枣 肠道菌群酵解 酚类物质 氨基甲酸乙酯 抗氧化活性 1 Introduction 1 Chinese jujube ( Ziziphus jujuba Mill.) belongs to the Rhamnaceae family, and is native to China. It has been domesticated in China for more than 7000 years, with current production rates at approximately 6.25 million tons of dry fruit per year ( Huang et al., 2016 Huang et al., 2016 ). The jujube is a famous traditional resource with numerous biological activity ( Pahuja et al., 2011 Pahuja et al., 2011 ; Plastina et al., 2012 Plastina et al., 2012 ) and is highly respected for its unique flavor and nutritional content, such as flavonoids, amino acids, polysaccharides, triterpene acids, and phenolic acids ( Chen JP et al., 2013 Chen JP et al., 2013 ; Du et al., 2013 Du et al., 2013 ). Jujube possesses various health benefits such as antioxidant activity ( Zhang et al., 2018 Zhang et al., 2018 ), anticancer properties ( Ji et al., 2020 Ji et al., 2020 ), hepatoprotective properties ( Li Y et al., 2018 Li Y et al., 2018 ), and anti-inflammatory properties ( Periasamy et al., 2020 Periasamy et al., 2020 ). Polyphenols are important secondary metabolites available in plants with potential antioxidant properties ( Ravisankar et al., 2019 Ravisankar et al., 2019 ; Zhang et al., 2019 Zhang et al., 2019 ; Zhu et al., 2019 Zhu et al., 2019 ; Luca et al., 2020 Luca et al., 2020 ). High dietary intake of polyphenol-rich fruits and vegetables is negatively correlated with the onset of diabetes ( Dinda et al., 2020 Dinda et al., 2020 ) and cardiometabolic disorders ( Mozaffarian and Wu, 2018 Mozaffarian and Wu, 2018 ). The intake of polyphenols prompts complex metabolic processes in the body; they form biologically active metabolites and decomposition products, and then interact with gut microbiota and microbial enzymes ( Rowland et al., 2018 Rowland et al., 2018 ; Su et al., 2020 Su et al., 2020 ). The bioactivity of dietary polyphenols is more dependent on bioavailability than on their parent compounds ( Cardona et al., 2013 Cardona et al., 2013 ; Kawabata et al., 2019 Kawabata et al., 2019 ). Oxidative stress (OS) plays an important role in the development of type 2 diabetes (T2D) ( Rocha et al., 2020 Rocha et al., 2020 ), non-alcoholic fatty liver disease (NAFLD) ( Podszun et al., 2020 Podszun et al., 2020 ), aging ( Luo et al., 2020 Luo et al., 2020 ), and cancer ( Kou et al., 2020 Kou et al., 2020 ). Ethyl carbamate (EC) is recognized as a Group 2A carcinogen by the International Agency for Research on Cancer (IARC). It is commonly present in fermented foods and various alcoholic beverages. Cellular metabolites of EC are associated with oxidative stress and resulting DNA damage, and can form covalent bonds with DNA, RNA, or proteins ( Zhao et al., 2013 Zhao et al., 2013 ). Vinyl carbamate is generated from the adducts and C-hydroxylation of EC and then converted to epoxides which interact with DNA ( Colombo et al., 2015 Colombo et al., 2015 ). Previous study has shown that EC treatment leads to increased production of reactive oxygen species (ROS) in human intestinal epithelial cells ( Chen et al., 2016b Chen et al., 2016b ). Although there have been some studies on the biological activity of jujube fruit ( Wang, 2011 Wang, 2011 ; Huang et al., 2017 Huang et al., 2017 ), the antioxidant activity of its metabolites after undergoing the gut microbiota fermentation (GMF) which is part of the digestive process has not been studied in detail. This knowledge is important to determine whether jujube fruit has the potential for significant antioxidant activity in the body when eaten. Therefore, in this study we aimed to investigate the antioxidant activity and biologically active components of jujube fruit after GMF and to evaluate whether compounds from jujube fruit could scavenge EC-induced ROS overproduction after GMF. In addition, we evaluated the effect of jujube fruit after GMF on mitochondrial dysfunction caused by EC treatment in vitro and revealed its possible mechanism. 2 Materials and methods 1 2.1 Chemicals and materials 2 Rhodamine 123 (Rh123), 2',7'-dichlorofluorescin diacetate (DCFH-DA), and naphthalene-2,3-dicarboxal-dehyde (NDA) were purchased from Molecular Probes, Inc. (Eugene, OR, USA). In addition, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Hoechst 33258 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chromatographic-grade (≥99.9%) acetonitrile and methanol were purchased from Aladdin, Inc. (Shanghai, China). The gallic acid, protocatechuic acid, catechol, catechin, p -hydroxybenzoic acid, chlorogenic acid, caffeic acid, epicatechin, p -coumaric acid, ferulic acid, rutin, ellagic acid, cinnamic acid, and quercetin used were of high-performance liquid chromatography (HPLC) grade (≥99%), and were purchased from Shanghai Yuanye Biotechnology Company (Shanghai, China). All reagents used were of analytical grade (≥99%) unless otherwise mentioned. We obtained fresh jujube fruit ( Zizyphus jujuba cv. Junzao) from Xinjiang Uygur Autonomous Region, China in October 2018, and screened it for uniform shape, size, and color. The initial average moisture content was 65.82% (wet basis) and the fruit was oven-dried to a constant weight at 105 ℃. 2.2 In vitro gut microbiota fermentation 2 The gastrointestinal digestion procedure was first carried out on the fresh jujube fruit according to a previously described method ( Bao et al., 2018 Bao et al., 2018 , 2019 2019 ). In short, 10 g of fresh jujube fruit was homogenized and then diluted with distilled water (10 mL). The sample was brought to pH 2.0 and blended with porcine pepsin (6000 U), followed by incubation for 2 h at 37 ℃ in a shaking water bath at 100 r/min simulating gastric digestion. Then, the mixture was adjusted to pH 6.0 and blended with pancreatin (20 mg) and bile salts (125 mg). Afterward, the pH was adjusted to 7.4 and the mixture was incubated at 37 ℃ for 2 h to simulate intestinal digestion. Thereafter, in vitro human GMF was conducted for gastrointestinal digested fresh jujube fruit homogenate following a previous method ( Gowd et al., 2018 Gowd et al., 2018 ). Three healthy diet and antibiotic-free donors provided stool samples. The stool samples were diluted to a concentration of 1% (0.01 g/mL) using 0.2 mol/L of carbonate phosphate buffer (pH 5.5), followed by filtration using a 1-mm sieve. Then, the digested jujube fruit homogenate was mixed with stool solution and kept in an anaerobic environment with oxygen-free gas (85% N 2 , 10% CO 2 , and 5% H 2 ; volume fraction), with constant stirring at different time intervals (0, 2, 6, 12, 24, and 48 h) at 37 ℃. The supernatant was collected, filtered using 0.22 μm Millipore filter (Bedford, MA, USA), and finally preserved at -80 ℃ for experimental analysis. 2.3 Estimation of total phenolic, flavonoid, sugar, and reducing sugar contents 2 A Folin-Ciocalteu assay was conducted to determine the total phenolic contents of the jujube fruit ( Singleton and Rossi, 1965 Singleton and Rossi, 1965 ), and values were expressed as milligram gallic acid equivalent (GAE) per gram of fresh weight (FW). To determine the total flavonoid contents of the fruit, we applied a colorimetric method ( Chang et al., 2002 Chang et al., 2002 ), and expressed values as milligram rutin equivalent (RE) per gram of FW. However, the total sugars and total reducing sugars were determined according to the Association of Official Analytical Chemists (AOAC) standard procedures ( Miller, 1959 Miller, 1959 ; AOAC, 1995 AOAC, 1995 ). 2.4 Phenolic compound analysis by HPLC 2 The phenolic compounds of the jujube fruit were measured by a Dionex Ultimate 3000 HPLC system (ThermoFisher Scientific, Waltham, MA, USA) which was equipped with a diode array detector ( Li YT et al., 2018 Li YT et al., 2018 ). The separations were operated on a C18 column (250 mm×4.60 mm, 5 μm), and the flow rate was fixed at 0.8 mL/min. In addition, the column temperature was set at 30 ℃, and injection volume of jujube samples was set at 20 μL. A mobile phase gradient (composed of two solvents such as 1.5% of aqueous formic acid solution (A) and methanol (B)) was used for the following linear gradient program: 0–15 min, 15% to 25% phase B; 15‒25 min, 25% phase B; 25‒60 min, 25% to 69% phase B; 60‒62 min, 69% to 100% phase B; 62‒67 min, 100% phase B; 67‒70 min, 100% to 15% phase B. Absorbance was recorded at 280 and 360 nm. 2.5 In vitro antioxidant assay 2 2.5.1　FRAP assay 3 The antioxidant capacity of jujube samples was calculated using a ferric reducing antioxidant power (FRAP) assay ( Chen W et al., 2013 Chen W et al., 2013 ). In brief, 50 μL of the fermented jujube was incubated for 30 min with 150 μL of FRAP solution at 37 ℃. The absorbance was measured at 593 nm and the values were represented as milligram vitamin C equivalent per gram of FW. 2.5.2　ABTS assay 3 The 2,2'-azinobis-‍(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical scavenging activity was calculated following a previously established method ( Li et al., 2019 Li et al., 2019 ). Fermented jujube (50 μL) was incubated for 6 min in the dark with 150 μL of ABTS •+ solution. The absorbance was measured at 734 nm and the values were expressed as half maximal inhibitory concentration (IC 50 ). 2.6 Cell culture 2 A human intestinal epithelial cell line (Caco-2) was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) and supplemented with fetal bovine serum (FBS; 10% (volume fraction)), streptomycin (0.1 mg/mL), and penicillin (100 U/mL). The cells were maintained at 37 ℃ with 5% CO 2 in an incubator ( Chen et al., 2016a Chen et al., 2016a ). 2.7 Cell viability assay 2 First, the Caco-2 cells were seeded into a 96-well plate with a density of 6×10 3 cells/well and pretreated with different concentrations of digested jujube fruit (0.25, 0.50, 1.00, or 2.00 mg/mL) for 24 h. After incubation, the cells were treated with 60 mmol/L of EC for another 24 h followed by washing with phosphate-buffered saline (PBS), and then incubated with MTT (0.5 mg/mL) for 4 h. The absorbance was recorded at 490 nm using a Tecan Infinite M200 microplate reader (Grodig, Austria) ( Hu et al., 2018 Hu et al., 2018 ). 2.8 Nuclear staining analysis 2 Hoechst 33258 stain solution was used for nuclear staining as in a previously published report ( Chen et al., 2017 Chen et al., 2017 ). Briefly, as described in Section 2.7, the cells were treated with jujube fruit samples and EC. Next, the treated cells were collected, followed by staining with Hoechst 33258 (10 μmol/L) at 37 ℃ for 30 min. Then, the cells were visualized under a fluorescence microscope. 2.9 Determination of intracellular ROS 2 A fluorescent probe dye, DCFH-DA, was used to determine intracellular ROS generation following a previous study ( Chen et al., 2016b Chen et al., 2016b ). Then, the Caco-2 cells were seeded into a 24-well plate (density 2.5×10 4 cells/well). The samples and EC were incubated with cells as described in Section 2.7. After that, 10 μmol/L of DCFH-DA was added to the treated cells for 30 min at 37 ℃. A fluorescence microscope was used to evaluate cellular fluorescence. The results were calculated from the fluorescence images with ImageProPlus6.0 (Media Cybernetics, Inc., Singapore). 2.10 Determination of cellular GSH 2 Cellular glutathione (GSH) content was analyzed using NDA as described previously ( Chen et al., 2014 Chen et al., 2014 ). The samples and EC were incubated with cells as described in Section 2.9. Then, 50 μmol/L of NDA was incubated with cells for 30 min at 37 ℃. Afterwards, the cells were visualized under a fluorescence microscope. The values were calculated as described in Section 2.9. 2.11 Determination of mitochondrial membrane potential 2 The same treatment conditions were adopted as detailed in Section 2.9. The cells were then incubated with 10 μg/mL of Rh123 for 30 min at 37 ℃ ( Gowd et al., 2019 Gowd et al., 2019 ). The values were calculated as mean Rh123 fluorescence intensity, as described in Section 2.9. 2.12 Statistical analysis 2 All experimental data were analyzed as mean±standard deviation (SD) using SPSS (Version 22.0). One-way analysis of variance (ANOVA) with Tukey's post-hoc analysis was done for multiple comparison testing. P < 0.05 was considered statistically significant. 3 Results 1 3.1 Determination of phenolic components in fermented jujube fruit 2 We analyzed phenolic, flavonoid, and sugar contents in fermented jujube fruit (0‒48 h fermentation). As shown in Fig. 1 Fig. 1 , total phenolic compounds were sharply raised at 2 h (5.91 mg GAE/g FW) and gradually declined until 48 h (4.64 mg GAE/g FW) ( P < 0.05; Fig. 1 Fig. 1 a). The total flavonoid contents reached their maximum at 2 h (0.34 mg RE/g FW) of GMF, and then dropped steeply, reaching a minimum at 24 h (0.13 mg RE/g FW) and remaining unchanged until 48 h (0.13 mg RE/g FW) ( Fig. 1 Fig. 1 b). The total sugar content (TSC) and reducing sugar content (RSC) were also detected throughout the GMF process. TSC declined gradually, from 417.06 to 205.82 mg glucose equivalent (GluE)/g FW ( P < 0.05; Fig. 1 Fig. 1 c); however, RSC increased significantly in the first 6 h ( P < 0.05), and then increased gradually, reaching a maximum at 24 h (124.65 mg GluE/g FW) and remaining unchanged until 48 h (122.84 mg GluE/g FW) ( P > 0.05; Fig. 1 Fig. 1 d). Taken together, these results showed that gut microbiota can decompose polyphenols and flavonoids in jujube. They also can decompose TSC and increase RSC. 3.2 Identification of phenolics of jujube after gut microbiota fermentation 2 Phenolic compounds of fermented jujube fruit (0‒48 h fermentation) were detected by HPLC ( Table 1 Table 1 and Fig. S1). We monitored the changes of 13 representative polyphenol compounds in jujube. Protocatechuic acid, catechol, catechin, chlorogenic acid, p -coumaric acid, ferulic acid, ellagic acid, and cinnamic acid reached their maximum contents at 2 h of GMF, and then stayed at a low level with the prolongation of GMF. p -Hydroxybenzoic acid and caffeic acid had the highest contents when GMF reached 24 h. The contents of epicatechin, rutin, and quercetin decreased with prolonged GMF. Epicatechin, rutin, and cinnamic acid were not detected after 12 h of GMF, indicating that these three compounds were either easily metabolized and decomposed by gut microbiota, or that they were structurally unstable. Catechol content remained at a high level compared to other polyphenol compounds after digested jujube fruit underwent GMF. Therefore, we believe that the antioxidant capacity of digested jujube fruit may be dominated by catechol. In a word, GMF could somewhat increase the content of phenolic compounds in jujube fruit, while catechol had the highest content in digested jujube fruit after GMF. Data were expressed as mean±standard deviation ( n =3). Values in the same column with different superscript letters are significantly different at P < 0.05 according to the Duncan test. FW, fresh weight. Time (h) 2 Protocatechuic acid 2 Catechol 2 Catechin 3 p- Hydroxybenzoic acid 2 Chlorogenic acid Caffeic acid 0 2 5.28±0.13 e 2 24.49±1.44 d 2 3.48±0.32 c 3 2.35±0.22 b 2 13.11±0.58 d 1.46±0.19 bc 2 2 6.13±0.25 f 2 27.89±2.32 e 2 4.05±0.05 d 3 2.63±0.09 b 2 15.42±1.52 e 1.50±0.23 bc 6 2 2.87±0.01 c 2 10.64±0.11 b 2 2.03±0.20 b 3 1.87±0.12 a 2 8.76±0.15 bc 1.28±0.07 ab 12 2 1.74±0.11 a 2 7.38±0.17 a 2 1.38±0.10 a 3 2.28±0.28 b 2 7.97±0.46 b 1.18±0.03 a 24 2 2.27±0.07 b 2 14.04±0.54 c 2 2.03±0.22 b 3 5.11±0.31 d 2 9.99±0.23 c 1.68±0.02 c 48 2 4.55±0.29 d 2 9.93±0.47 b 2 1.67±0.06 a 3 4.63±0.12 c 2 4.33±0.02 a 1.33±0.01 ab Time (h) Epicatechin 2 p- Coumaric acid 2 Ferulic acid 2 Rutin Ellagic acid 2 Cinnamic acid 2 Quercetin 0 5.84±0.16 c 2 0.86±0.05 d 2 2.25±0.22 c 2 0.83±0.12 c 1.24±0.12 a 2 0.52±0.01 b 2 2.09±0.09 f 2 5.31±0.27 b 2 0.95±0.10 d 2 3.00±0.35 d 2 0.26±0.04 a 1.82±0.07 b 2 0.58±0.04 c 2 0.77±0.03 c 6 3.25±0.20 a 2 0.48±0.05 c 2 1.35±0.14 b 2 0.44±0.03 b 1.75±0.18 b 2 0.40±0.04 a 2 1.24±0.04 e 12 2 0.31±0.01 b 2 0.56±0.06 a 2 1.23±0.01 a 2 2 0.88±0.01 d 24 2 2 0.66±0.01 a 2 1.70±0.02 b 2 2 0.21±0.01 b 48 2 0.03±0.01 a 2 0.50±0.03 a 2 1.64±0.10 b 2 2 0.02±0.00 a 3.3 Determination of antioxidant activity in fermented jujube fruit 2 The antioxidant activity of jujube (0‒48 h fermentation) was further studied using in vitro antioxidant assays (ABTS and FRAP). According to ABTS assay ( Fig. 2 Fig. 2 a), the inhibitory effect of fermented jujube increased significantly with the extension of GMF, exhibiting the best inhibitory activity (0.0139 mg/mL) at 2 h, at which point inhibitory activity reduced steeply. The IC 50 values were 0.0150, 0.0139, 0.0160, 0.0197, 0.0213, and 0.0209 mg/mL. The antioxidant activity of fermented jujube was further assessed by FRAP assay ( Fig. 2 Fig. 2 b). The ferric ion reduction ability of samples significantly increased after GMF at 2 h, then gradually decreased until 24 h, and returned to the initial level at 48 h. Overall, gut microbiota can enhance the free-radical scavenging capacity of fermented jujube in a time-dependent manner, but only temporarily improves ferric reducing ability, and then returns to normal levels. 3.4 Inhibitory effects of digested jujube fruit on EC-induced toxicity and DNA damage 2 EC-induced toxicity has been found in both in vitro cellular and in vivo animal studies ( Chun et al., 2013 Chun et al., 2013 ). We collected metabolites of fermented jujube under GMF treatment at 48 h and employed the Caco-2 cell model to further identify differences in the effectiveness of jujube fruit against EC impairment based on antioxidant capacity. Different fermented jujube fruit samples (0.25, 0.50, 1.00, and 2.00 mg/mL) did not induce any toxicity to Caco-2 cells ( P > 0.05; Fig. 3 Fig. 3 a) and cell viability was significantly decreased by treatment with EC ( P < 0.05; Fig. 3 Fig. 3 b). Caco-2 cells pretreated with 0.50 mg/mL samples of fermented jujube fruit with 60 mmol/L EC showed increased viability from 63% to 70%, while 1.00 mg/mL of fermented jujube fruit samples increased viability to 74% ( P < 0.001; Fig. 3 Fig. 3 b). Next, we investigated the efficacy of metabolites of fermented jujube (0.25, 0.50, and 1.00 mg/mL) against EC-treated DNA damage in Caco-2 cells. In the EC 60 mmol/L group, a relatively large number of cell nuclei were visible with bright blue dots, indicating that DNA was affected by nuclear fragmentation and/or chromatin condensation. However, cells treated with 1.00 mg/mL of fermented jujube samples for 24 h showed fewer cell nuclei with dark blue than the EC 60 mmol/L group ( P < 0.05), which was almost the same as the control ( Fig. 3 Fig. 3 c). Taken together, metabolites of fermented jujube at 0.50 or 1.00 mg/mL concentration under GMF can enhance cell viability and reduce cytotoxicity caused by EC. 3.5 Mitigation capacity of digested jujube fruit in EC-induced ROS generation 2 We further determined the antioxidant capacity of jujube after GMF in Caco-2 cells. According to Figs. 4a 4a and 4b 4b , the fluorescence intensity of the EC 60 mmol/L group was increased by 149% over the control group (normalized to 100%) ( P < 0.001). However, in fermented jujube fruit samples (0.25, 0.50, or 1.00 mg/mL), fluorescence intensity was lessened in a dose-dependent fashion ( P < 0.05) compared to the EC 60 mmol/L group ( Fig. 4 Fig. 4 b). Fermented jujube fruit samples at a concentration of 1.00 mg/mL showed better ROS scavenging activity (116%). The fluorescence intensities of fermented jujube sample groups at 0.25, 0.50, and 1.00 mg/mL were 130% ( P < 0.01), 117% ( P < 0.001), and 116% ( P < 0.001), respectively. Thus, jujube fruit fermented by gut microbiota could protect against EC-treated oxidative stress by hindering ROS overproduction. 3.6 Amelioration of EC-induced glutathione reduction by fermented jujube fruit 2 Since fermented jujube samples could effectively scavenge ROS induced by EC, we speculated that fermented jujube samples could also influence EC-treated GSH depletion. According to Figs. 4c 4c and 4d 4d , a marked reduction of fluorescence intensity was noticed in the EC 60 mmol/L group (49% of control) ( P < 0.001), and cells supplemented with fermented jujube at three concentrations (0.25, 0.50 and 1.00 mg/mL) significantly ameliorated cellular GSH depletion in a concentration-dependent fashion. The fluorescence intensities of fermented jujube sample groups at 0.25, 0.50, and 1.00 mg/mL were 59% ( P < 0.05), 72% ( P < 0.001), and 78% ( P < 0.001), respectively. Thus, we found that jujube fruit fermented by gut microbiota possessed potential cellular antioxidant activity. 3.7 Effect of fermented jujube fruit on EC-induced mitochondrial membrane impairment 2 ROS is mainly produced by mitochondria and affects mitochondrial metabolism ( Reddy, 2006 Reddy, 2006 ). Hence, we analyzed the function of fermented jujube samples on the mitochondrial membrane in the presence of EC. As shown in Figs. 5a 5a and 5b 5b , the fluorescence intensity of the EC 60 mmol/L group decreased to 71% of control, showing that exposure to EC leads to the reduction of mitochondrial membrane potential (MMP). Then, we evaluated the ameliorative effect of fermented jujube samples against EC-treated MMP collapse. All samples significantly augmented MMP of Caco-2 cells in a concentration-dependent fashion (0.25, 0.50, and 1.00 mg/mL). The fluorescence intensities of fermented jujube samples at 0.25, 0.50, and 1.00 mg/mL were 84% ( P < 0.001), 85% ( P < 0.001), and 92% ( P < 0.001), respectively. Taken together, jujube fruit fermented by gut microbiota has potential against EC-induced mitochondrial dysfunction. 4 Discussion 1 Jujube fruit is widely used in traditional Chinese medicine because of its high nutritional value and various pharmacological effects ( Guo et al., 2009 Guo et al., 2009 ). We have earlier reported the biologically active ingredients and antioxidant properties of dried jujube fruit ( Bao et al., 2021 Bao et al., 2021 ). However, knowledge of gastrointestinal and gut microbiota modifications, along with bioavailability and their impact on bioactivity, is lacking due to the many analytical challenges faced when analyzing jujube fruit. This study aimed to investigate the main active ingredients of jujube fruit after GMF and their mechanisms related to antioxidant activity. ABTS •+ is commonly used to assess free radical scavenging ability ( Gu et al., 2019 Gu et al., 2019 ). Thus, this study compared the antioxidant capacity of jujube fruit after GMF through ABTS •+ assay. Using traditional in vitro experiments to detect the contents of phenolics, flavonoids, and sugar, as well as the free radical scavenging ability of jujube fruit after GMF, we observed a reduction of all of these in vitro, except that the content of reducing sugars increased. We speculate that total sugar is decomposed into reducing sugars due to the metabolism of intestinal flora. In another study on vegetables, correlation statistical analysis between the ABTS •+ value and the phenolic contents indicated significant correlations ( Dudonné et al., 2009 Dudonné et al., 2009 ). Jujube still possessed considerable antioxidant activity (FRAP) after GMF compared with dried jujube fruit ( Bao et al., 2021 Bao et al., 2021 ). At the same time, our findings are consistent with a previous published report ( Choi et al., 2011 Choi et al., 2011 ), in which FRAP is not significantly correlated with the duration of GMF that jujube fruit undergoes. These results indicate that measurements of total polyphenol content and free radical scavenging potential cannot be considered accurate in terms of the assumed beneficial health effects if the effects of digestion and fermentation processes in the gastrointestinal tract are not considered. In GMF, dietary polyphenols can act as substrates for various enzymes in the small intestine and stomach when exposed to specific pH conditions ( Singh et al., 2017 Singh et al., 2017 ). The polyphenols in the colon may undergo further transformations, and the enzymes of the intestinal flora break down the complex polyphenolic structure into smaller units. Bacterial enzymes can catalyze many reactions, including hydrolysis, demethylation, dehydroxylation, ring cleavage, and decarboxylation ( Fraga et al., 2019 Fraga et al., 2019 ). A growing body of work involving intervention in vivo and epidemiological study of dietary polyphenols has provided new evidence on a variety of health-promoting activities. These data have been examined from in vitro data records, including anti-inflammatory ( Yahfoufi et al., 2018 Yahfoufi et al., 2018 ), antioxidant ( Mojzer et al., 2016 Mojzer et al., 2016 ), anticancer ( Niedzwiecki et al., 2016 Niedzwiecki et al., 2016 ), antidiabetic ( Cao et al., 2019 Cao et al., 2019 ), and neuroprotective potential ( Spagnuolo et al., 2016 Spagnuolo et al., 2016 ), suggesting a positive correlation between the consumption of polyphenol-rich foods and lower risk of chronic diseases. In order to explore the changes of polyphenol contents in jujube fruit under GMF, we quantified polyphenol compounds in fermented jujube and found that the contents of most polyphenols increased briefly after GMF and then decreased, which may be due to the decomposition of jujube fruit by gut microbiota; however, the instability of the digestive system may result in a decrease in polyphenol contents. Our previous study along with others indicates that jujube contains abundant phenolic compounds such as rutin, chlorogenic acid, catechol, catechin, p- hydroxybenzoic acid, caffeic acid, and ferulic acid ( Gao et al., 2013 Gao et al., 2013 ; Zhao et al., 2014 Zhao et al., 2014 ; Bao et al., 2021 Bao et al., 2021 ). Rutin (142.05 to 292.33 mg/kg dry weight) is the predominant phenolic compound in dried and fresh jujube samples ( Bao et al., 2021 Bao et al., 2021 ). However, the rutin content in jujube fruit ranges from 0.26 to 0.83 mg/kg FW after GMF for 0‒6 h, and is undetectable after 12‒48 h of GMF. This could be due to the gastrointestinal digestion procedure performed before GMF, which caused the degradation of rutin ( Siracusa et al., 2011 Siracusa et al., 2011 ). Fermented jujube fruit still possesses considerable in vitro antioxidant activity, possibly because of the presence of multiple phenolic substances such as catechol, protocatechuic acid, p- hydroxybenzoic acid, and chlorogenic acid. We found the highest content of catechol in jujube fruit after GMF. It has also been reported that catechol has antioxidant activity and has shown beneficial effects in diseases such as diabetes ( Cremonini et al., 2019 Cremonini et al., 2019 ), Alzheimer disease ( Kim et al., 2020 Kim et al., 2020 ), and obesity ( Wang et al., 2017 Wang et al., 2017 ). Some in vitro and in vivo studies have revealed EC-induced toxicity ( Field and Lang, 1988 Field and Lang, 1988 ; Cui et al., 2016 Cui et al., 2016 ). Additionally, in the present study, we used Caco-2 cells to explore the effectiveness of jujube fruit against EC-induced cytotoxicity and DNA damage after GMF. Although the contents of phenolics and flavonoids in fermented jujube are significantly reduced, the fruit still has a certain amount of concentration-dependent activity in resisting EC-induced DNA damage and improving cell viability. This indicates that the metabolites of jujube fruit fermented by gut microbiota may have biological activity against DNA damage and improve cell growth. Disturbance of the redox balance between ROS and antioxidants in cells is related to cytotoxicity ( Tseng et al., 2017 Tseng et al., 2017 ). Jujube can reduce the overproduction of ROS induced by EC after GMF and shows a concentration-dependent effect. It has been reported that phenolic compounds can inhibit intracellular OS induced by exogenous substances ( Lin et al., 2016 Lin et al., 2016 ). Thus, the inhibitory effect of jujube on EC-treated oxidative stress after GMF may be associated with the phenolic content. GSH is the core of the intracellular antioxidant defense system ( Lv et al., 2019 Lv et al., 2019 ), and can scavenge ROS, thereby helping to control redox homeostasis ( Bansal and Simon, 2018 Bansal and Simon, 2018 ). Supplementing the diet with fermented jujube fruit significantly restores GSH, thereby enhancing the endogenous antioxidant defense mechanism against OS. ROS is generated primarily by mitochondria, affecting mitochondrial metabolism ( Cadenas, 2018 Cadenas, 2018 ). The maintenance of MMP is necessary for normal cell function and has a significant function in maintaining cell ROS homeostasis. Overproduction and accumulation of ROS are also related to mitochondrial dysfunction and MMP collapse ( Murphy, 2016 Murphy, 2016 ). Here, we report that the metabolites of jujube that has undergone GMF have the potential to resist EC-induced, ROS-related mitochondrial dysfunction, preventing oxidative stress induced by mitochondrial dysfunction. Consequently, this study uncovers some important insights about the activity of bioactive components of jujube after GMF, provides a basis for further research on the bioactive components of jujube in vivo, and helps to clarify the mechanism of action in GMF. 5 Conclusions 1 This study unveiled the effects of GMF on the antioxidant capacity of bioactive substances obtained from jujube fruit. As far as we know, there are several studies on the polyphenols of jujube and their biological activity, but none on the antioxidant activity of polyphenols in jujube fruit after GMF. The addition of jujube fruit samples significantly increases the cell viability of Caco-2 cells and reduces DNA damage after GMF; it also lessens the excessive ROS production caused by EC and significantly restores GSH levels and mitochondrial membrane potential in Caco-2 cells. In short, jujube polyphenols show potent antioxidant activity after GMF, and jujube can thus be considered a potential ingredient for development of functional foods. References AOAC , 1995 . Official Methods of Analysis , 16th Ed. Association of Official Analytical Chemists , Arlington, USA . Official Methods of Analysis Bansal A , Simon MC , 2018 . Glutathione metabolism in cancer progression and treatment resistance . J Cell Biol , 217 ( 7 ): 2291 - 2298 . https://doi.org/10.1083/jcb.201804161 https://doi.org/10.1083/jcb.201804161 Glutathione metabolism in cancer progression and treatment resistance Bao T , Li YT , Xie JH , et al. , 2018 . Systematic evaluation of bioactive components and antioxidant capacity of some new and common bayberry cultivars using an in vitro gastrointestinal digestion method . Food Res Int , 103 : 326 - 334 . https://doi.org/10.1016/j.foodres.2017.10.062 https://doi.org/10.1016/j.foodres.2017.10.062 Systematic evaluation of bioactive components and antioxidant capacity of some new and common bayberry cultivars using an in vitro gastrointestinal digestion method Bao T , Li YT , Xie JH , et al. , 2019 . Systematic evaluation of polyphenols composition and antioxidant activity of mulberry cultivars subjected to gastrointestinal digestion and gut microbiota fermentation . J Funct Foods , 58 : 338 - 349 . https://doi.org/10.1016/j.jff.2019.05.017 https://doi.org/10.1016/j.jff.2019.05.017 Systematic evaluation of polyphenols composition and antioxidant activity of mulberry cultivars subjected to gastrointestinal digestion and gut microbiota fermentation Bao T , Hao X , Shishir MRI , et al. , 2021 . Cold plasma: an emerging pretreatment technology for the drying of jujube slices . Food Chem , 337 : 127783 . https://doi.org/10.1016/j.foodchem.2020.127783 https://doi.org/10.1016/j.foodchem.2020.127783 Cold plasma: an emerging pretreatment technology for the drying of jujube slices Cadenas S , 2018 . Mitochondrial uncoupling, ROS generation and cardioprotection . Biochim Biophys Acta Bioenerg , 1859 ( 9 ): 940 - 950 . https://doi.org/10.1016/j.bbabio.2018.05.019 https://doi.org/10.1016/j.bbabio.2018.05.019 Mitochondrial uncoupling, ROS generation and cardioprotection Cao H , Ou JY , Chen L , et al. , 2019 . Dietary polyphenols and type 2 diabetes: human study and clinical trial . Crit Rev Food Sci Nutr , 59 ( 20 ): 3371 - 3379 . https://doi.org/10.1080/10408398.2018.1492900 https://doi.org/10.1080/10408398.2018.1492900 Dietary polyphenols and type 2 diabetes: human study and clinical trial Cardona F , Andrés-Lacueva C , Tulipani S , et al. , 2013 . Benefits of polyphenols on gut microbiota and implications in human health . J Nutr Biochem , 24 ( 8 ): 1415 - 1422 . https://doi.org/10.1016/j.jnutbio.2013.05.001 https://doi.org/10.1016/j.jnutbio.2013.05.001 Benefits of polyphenols on gut microbiota and implications in human health Chang CC , Yang MH , Wen HM , et al. , 2002 . Estimation of total flavonoid content in propolis by two complementary colorimetric methods . J Food Drug Anal , 10 ( 3 ): 178 - 182 . https://doi.org/10.38212/2224-6614.2748 https://doi.org/10.38212/2224-6614.2748 Estimation of total flavonoid content in propolis by two complementary colorimetric methods Chen JP , Li ZG , Maiwulanjiang M , et al. , 2013 . Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages . J Agric Food Chem , 61 ( 30 ): 7315 - 7324 . https://doi.org/10.1021/jf402379u https://doi.org/10.1021/jf402379u Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages Chen W , Feng LN , Shen Y , et al. , 2013 . Myricitrin inhibits acrylamide-mediated cytotoxicity in human Caco-2 cells by preventing oxidative stress . Biomed Res Int , 2013 : 724183 . https://doi.org/10.1155/2013/724183 https://doi.org/10.1155/2013/724183 Myricitrin inhibits acrylamide-mediated cytotoxicity in human Caco-2 cells by preventing oxidative stress Chen W , Shen Y , Su HM , et al. , 2014 . Hispidin derived from Phellinus linteus affords protection against acrylamide-induced oxidative stress in Caco-2 cells . Chem Biol Interact , 219 : 83 - 89 . https://doi.org/10.1016/j.cbi.2014.05.010 https://doi.org/10.1016/j.cbi.2014.05.010 Hispidin derived from Phellinus linteus affords protection against acrylamide-induced oxidative stress in Caco-2 cells Chen W , Su HM , Xu Y , et al. , 2016a . Protective effect of wild raspberry ( Rubus hirsutus Thunb .) extract against acrylamide-induced oxidative damage is potentiated after simulated gastrointestinal digestion. Food Chem , 196 : 943 - 952 . https://doi.org/10.1016/j.foodchem.2015.10.024 https://doi.org/10.1016/j.foodchem.2015.10.024 Protective effect of wild raspberry (Rubus hirsutus Thunb Chen W , Xu Y , Zhang LX , et al. , 2016b . Blackberry subjected to in vitro gastrointestinal digestion affords protection against ethyl carbamate-induced cytotoxicity . Food Chem , 212 : 620 - 627 . https://doi.org/10.1016/j.foodchem.2016.06.031 https://doi.org/10.1016/j.foodchem.2016.06.031 Blackberry subjected to in vitro gastrointestinal digestion affords protection against ethyl carbamate-induced cytotoxicity Chen W , Su HM , Xu Y , et al. , 2017 . In vitro gastrointestinal digestion promotes the protective effect of blackberry extract against acrylamide-induced oxidative stress . Sci Rep , 7 : 40514 . https://doi.org/10.1038/srep40514 https://doi.org/10.1038/srep40514 In vitro gastrointestinal digestion promotes the protective effect of blackberry extract against acrylamide-induced oxidative stress Choi SH , Ahn JB , Kozukue N , et al. , 2011 . Distribution of free amino acids, flavonoids, total phenolics, and antioxidative activities of jujube ( Ziziphus jujuba ) fruits and seeds harvested from plants grown in Korea . J Agric Food Chem , 59 ( 12 ): 6594 - 6604 . https://doi.org/10.1021/jf200371r https://doi.org/10.1021/jf200371r Distribution of free amino acids, flavonoids, total phenolics, and antioxidative activities of jujube (Ziziphus jujuba) fruits and seeds harvested from plants grown in Korea Chun SH , Cha YN , Kim C , 2013 . Urethane increases reactive oxygen species and activates extracellular signal-regulated kinase in RAW 264 .7 macrophages and A549 lung epithelial cells. Arch Pharm Res , 36 ( 6 ): 775 - 782 . https://doi.org/10.1007/s12272-013-0104-8 https://doi.org/10.1007/s12272-013-0104-8 Urethane increases reactive oxygen species and activates extracellular signal-regulated kinase in RAW Colombo NBR , Rangel MP , Martins V , et al. , 2015 . Caryocar brasiliense camb protects against genomic and oxidative damage in urethane-induced lung carcinogenesis . Braz J Med Biol Res , 48 ( 9 ): 852 - 862 . https://doi.org/10.1590/1414-431x20154467 https://doi.org/10.1590/1414-431x20154467 Caryocar brasiliense camb protects against genomic and oxidative damage in urethane-induced lung carcinogenesis Cremonini E , Fraga CG , Oteiza PI , 2019 . (-)-Epicatechin in the control of glucose homeostasis: involvement of redox-regulated mechanisms . Free Radic Biol Med , 130 : 478 - 488 . https://doi.org/10.1016/j.freeradbiomed.2018.11.010 https://doi.org/10.1016/j.freeradbiomed.2018.11.010 (-)-Epicatechin in the control of glucose homeostasis: involvement of redox-regulated mechanisms Cui X , Wang JY , Qiu NN , et al. , 2016 . In vitro toxicological evaluation of ethyl carbamate in human HepG2 cells . Toxicol Res (Camb) , 5 ( 2 ): 697 - 702 . https://doi.org/10.1039/c5tx00453e https://doi.org/10.1039/c5tx00453e In vitro toxicological evaluation of ethyl carbamate in human HepG2 cells Dinda B , Dinda M , Roy A , et al. , 2020 . Dietary plant flavonoids in prevention of obesity and diabetes . Adv Protein Chem Struct Biol , 120 : 159 - 235 . https://doi.org/10.1016/bs.apcsb.2019.08.006 https://doi.org/10.1016/bs.apcsb.2019.08.006 Dietary plant flavonoids in prevention of obesity and diabetes Du LJ , Gao QH , Ji XL , et al. , . 2013 . Comparison of flavonoids, phenolic acids, and antioxidant activity of explosion-puffed and sun-dried jujubes (Ziziphus jujuba Mill.) . J Agric Food Chem , 61 ( 48 ): 11840 - 11847 . https://doi.org/10.1021/jf401744c https://doi.org/10.1021/jf401744c Comparison of flavonoids, phenolic acids, and antioxidant activity of explosion-puffed and sun-dried jujubes (Ziziphus jujuba Mill.) Dudonné S , Vitrac X , Coutière P , et al. , 2009 . Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays . J Agric Food Chem , 57 ( 5 ): 1768 - 1774 . https://doi.org/10.1021/jf803011r https://doi.org/10.1021/jf803011r Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays Field KJ , Lang CM , 1988 . Hazards of urethane (ethyl carbamate): a review of the literature . Lab Anim , 22 ( 3 ): 255 - 262 . https://doi.org/10.1258/002367788780746331 https://doi.org/10.1258/002367788780746331 Hazards of urethane (ethyl carbamate): a review of the literature Fraga CG , Croft KD , Kennedy DO , et al. , 2019 . The effects of polyphenols and other bioactives on human health . Food Funct , 10 ( 2 ): 514 - 528 . https://doi.org/10.1039/c8fo01997e https://doi.org/10.1039/c8fo01997e The effects of polyphenols and other bioactives on human health Gao QH , Wu CS , Wang M , 2013 . The jujube ( Ziziphus jujuba Mill.) fruit: a review of current knowledge of fruit composition and health benefits . J Agric Food Chem , 61 ( 14 ): 3351 - 3363 . https://doi.org/10.1021/jf4007032 https://doi.org/10.1021/jf4007032 The jujube ( Ziziphus jujuba Mill.) fruit: a review of current knowledge of fruit composition and health benefits Gowd V , Bao T , Wang LL , et al. , 2018 . Antioxidant and antidiabetic activity of blackberry after gastrointestinal digestion and human gut microbiota fermentation . Food Chem , 269 : 618 - 627 . https://doi.org/10.1016/j.foodchem.2018.07.020 https://doi.org/10.1016/j.foodchem.2018.07.020 Antioxidant and antidiabetic activity of blackberry after gastrointestinal digestion and human gut microbiota fermentation Gowd V , Bao T , Chen W , 2019 . Antioxidant potential and phenolic profile of blackberry anthocyanin extract followed by human gut microbiota fermentation . Food Res Int , 120 : 523 - 533 . https://doi.org/10.1016/j.foodres.2018.11.001 https://doi.org/10.1016/j.foodres.2018.11.001 Antioxidant potential and phenolic profile of blackberry anthocyanin extract followed by human gut microbiota fermentation Gu CH , Howell K , Dunshea FR , et al. , 2019 . LC-ESI-QTOF/MS characterisation of phenolic acids and flavonoids in polyphenol-rich fruits and vegetables and their potential antioxidant activities . Antioxidants (Basel) , 8 ( 9 ): 405 . https://doi.org/10.3390/antiox8090405 https://doi.org/10.3390/antiox8090405 LC-ESI-QTOF/MS characterisation of phenolic acids and flavonoids in polyphenol-rich fruits and vegetables and their potential antioxidant activities Guo S , Duan JA , Tang YP , et al. , 2009 . High-performance liquid chromatography—two wavelength detection of triterpenoid acids from the fruits of Ziziphus jujuba containing various cultivars in different regions and classification using chemometric analysis . J Pharm Biomed Anal , 49 ( 5 ): 1296 - 1302 . https://doi.org/10.1016/j.jpba.2009.03.006 https://doi.org/10.1016/j.jpba.2009.03.006 High-performance liquid chromatography—two wavelength detection of triterpenoid acids from the fruits of Ziziphus jujuba containing various cultivars in different regions and classification using chemometric analysis Hu DW , Xu Y , Xie JH , et al. , 2018 . Systematic evaluation of phenolic compounds and protective capacity of a new mulberry cultivar J33 against palmitic acid-induced lipotoxicity using a simulated digestion method . Food Chem , 258 : 43 - 50 . https://doi.org/10.1016/j.foodchem.2018.03.049 https://doi.org/10.1016/j.foodchem.2018.03.049 Systematic evaluation of phenolic compounds and protective capacity of a new mulberry cultivar J33 against palmitic acid-induced lipotoxicity using a simulated digestion method Huang J , Zhang CM , Zhao X , et al. , 2016 . The jujube genome provides insights into genome evolution and the domestication of sweetness/acidity taste in fruit trees . PLoS Genet , 12 ( 12 ): e1006433 . https://doi.org/10.1371/journal.pgen.1006433 https://doi.org/10.1371/journal.pgen.1006433 The jujube genome provides insights into genome evolution and the domestication of sweetness/acidity taste in fruit trees Huang WZ , Wang YJ , Jiang XY , et al. , 2017 . Protective effect of flavonoids from Ziziphus jujuba cv . Jinsixiaozao against acetaminophen-induced liver injury by inhibiting oxidative stress and inflammation in mice . Molecules , 22 ( 10 ): 1781 . https://doi.org/10.3390/molecules22101781 https://doi.org/10.3390/molecules22101781 Protective effect of flavonoids from Ziziphus jujubacv. Jinsixiaozao against acetaminophen-induced liver injury by inhibiting oxidative stress and inflammation in mice Ji XL , Hou CY , Gao YG , et al. , 2020 . Metagenomic analysis of gut microbiota modulatory effects of jujube ( Ziziphus jujuba Mill.) polysaccharides in a colorectal cancer mouse model . Food Funct , 11 ( 1 ): 163 - 173 . https://doi.org/10.1039/c9fo02171j https://doi.org/10.1039/c9fo02171j Metagenomic analysis of gut microbiota modulatory effects of jujube (Ziziphus jujuba Mill.) polysaccharides in a colorectal cancer mouse model Kawabata K , Yoshioka Y , Terao J , 2019 . Role of intestinal microbiota in the bioavailability and physiological functions of dietary polyphenols . Molecules , 24 ( 2 ): 370 . https://doi.org/10.3390/molecules24020370 https://doi.org/10.3390/molecules24020370 Role of intestinal microbiota in the bioavailability and physiological functions of dietary polyphenols Kim YJ , Sohn E , Kim JH , et al. , 2020 . Catechol-type flavonoids from the branches of Elaeagnus glabra f. oxyphylla exert antioxidant activity and an inhibitory effect on amyloid-β aggregation . Molecules , 25 ( 21 ): 4917 . https://doi.org/10.3390/molecules25214917 https://doi.org/10.3390/molecules25214917 Catechol-type flavonoids from the branches of Elaeagnus glabra f. oxyphylla exert antioxidant activity and an inhibitory effect on amyloid-β aggregation Kou LF , Sun R , Jiang XY , et al. , 2020 . Tumor microenvironment-responsive, multistaged liposome induces apoptosis and ferroptosis by amplifying oxidative stress for enhanced cancer therapy . ACS Appl Mater Interfaces , 12 ( 27 ): 30031 - 30043 . https://doi.org/10.1021/acsami.0c03564 https://doi.org/10.1021/acsami.0c03564 Tumor microenvironment-responsive, multistaged liposome induces apoptosis and ferroptosis by amplifying oxidative stress for enhanced cancer therapy Li Y , Guo S , Ren QJ , et al. , 2018 . Pharmacokinetic comparisons of multiple triterpenic acids from Jujubae fructus extract following oral delivery in normal and acute liver injury rats . Int J Mol Sci , 19 ( 7 ): 2047 . https://doi.org/10.3390/ijms19072047 https://doi.org/10.3390/ijms19072047 Pharmacokinetic comparisons of multiple triterpenic acids from Jujubae fructus extract following oral delivery in normal and acute liver injury rats Li YT , Bao T , Chen W , 2018 . Comparison of the protective effect of black and white mulberry against ethyl carbamate-induced cytotoxicity and oxidative damage . Food Chem , 243 : 65 - 73 . https://doi.org/10.1016/j.foodchem.2017.09.106 https://doi.org/10.1016/j.foodchem.2017.09.106 Comparison of the protective effect of black and white mulberry against ethyl carbamate-induced cytotoxicity and oxidative damage Li ZY , Bu QT , Wang J , et al. , 2019 . Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 20 ( 12 ): 983 - 994 . http://doi.org/10.1631/jzus.B1900344 http://doi.org/10.1631/jzus.B1900344 Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB Lin DR , Xiao MS , Zhao JJ , et al. , 2016 . An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes . Molecules , 21 ( 10 ): 1374 . https://doi.org/10.3390/molecules21101374 https://doi.org/10.3390/molecules21101374 An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes Luca SV , Macovei I , Bujor A , et al. , 2020 . Bioactivity of dietary polyphenols: the role of metabolites . Crit Rev Food Sci Nutr , 60 ( 4 ): 626 - 659 . https://doi.org/10.1080/10408398.2018.1546669 https://doi.org/10.1080/10408398.2018.1546669 Bioactivity of dietary polyphenols: the role of metabolites Luo J , Mills K , le Cessie S , et al. , 2020 . Ageing, age-related diseases and oxidative stress: what to do next? Ageing Res Rev , 57 : 100982 . https://doi.org/10.1016/j.arr.2019.100982 https://doi.org/10.1016/j.arr.2019.100982 Ageing, age-related diseases and oxidative stress: what to do next? Lv HH , Zhen CX , Liu JY , et al. , 2019 . Unraveling the potential role of glutathione in multiple forms of cell death in cancer therapy . Oxid Med Cell Longev , 2019 : 3150145 . https://doi.org/10.1155/2019/3150145 https://doi.org/10.1155/2019/3150145 Unraveling the potential role of glutathione in multiple forms of cell death in cancer therapy Miller GL , 1959 . Use of dinitrosalicylic acid reagent for determination of reducing sugar . Anal Chem , 31 ( 3 ): 426 - 428 . https://doi.org/10.1021/ac60147a030 https://doi.org/10.1021/ac60147a030 Use of dinitrosalicylic acid reagent for determination of reducing sugar Mojzer EB , Hrnčič MK , Škerget M , et al. , 2016 . Polyphenols: extraction methods, antioxidative action, bioavailability and anticarcinogenic effects . Molecules , 21 ( 7 ): 901 . https://doi.org/10.3390/molecules21070901 https://doi.org/10.3390/molecules21070901 Polyphenols: extraction methods, antioxidative action, bioavailability and anticarcinogenic effects Mozaffarian D , Wu JHY , 2018 . Flavonoids, dairy foods, and cardiovascular and metabolic health: a review of emerging biologic pathways . Circ Res , 122 ( 2 ): 369 - 384 . https://doi.org/10.1161/circresaha.117.309008 https://doi.org/10.1161/circresaha.117.309008 Flavonoids, dairy foods, and cardiovascular and metabolic health: a review of emerging biologic pathways Murphy MP , 2016 . Understanding and preventing mitochondrial oxidative damage . Biochem Soc Trans , 44 ( 5 ): 1219 - 1226 . https://doi.org/10.1042/bst20160108 https://doi.org/10.1042/bst20160108 Understanding and preventing mitochondrial oxidative damage Niedzwiecki A , Roomi MW , Kalinovsky T , et al. , 2016 . Anticancer efficacy of polyphenols and their combinations . Nutrients , 8 ( 9 ): 552 . https://doi.org/10.3390/nu8090552 https://doi.org/10.3390/nu8090552 Anticancer efficacy of polyphenols and their combinations Pahuja M , Mehla J , Reeta KH , et al. , 2011 . Hydroalcoholic extract of Zizyphus jujuba ameliorates seizures, oxidative stress, and cognitive impairment in experimental models of epilepsy in rats . Epilepsy Behav , 21 ( 4 ): 356 - 363 . https://doi.org/10.1016/j.yebeh.2011.05.013 https://doi.org/10.1016/j.yebeh.2011.05.013 Hydroalcoholic extract of Zizyphus jujuba ameliorates seizures, oxidative stress, and cognitive impairment in experimental models of epilepsy in rats Periasamy S , Wu WH , Chien SP , et al. , 2020 . Dietary Ziziphus jujuba fruit attenuates colitis-associated tumorigenesis: a pivotal role of the NF-κB/IL-6/JAK1/STAT3 pathway . Nutr Cancer , 72 ( 1 ): 120 - 132 . https://doi.org/10.1080/01635581.2019.1615515 https://doi.org/10.1080/01635581.2019.1615515 Dietary Ziziphus jujuba fruit attenuates colitis-associated tumorigenesis: a pivotal role of the NF-κB/IL-6/JAK1/STAT3 pathway Plastina P , Bonofiglio D , Vizza D , et al. , 2012 . Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells . J Ethnopharmacol , 140 ( 2 ): 325 - 332 . https://doi.org/10.1016/j.jep.2012.01.022 https://doi.org/10.1016/j.jep.2012.01.022 Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells Podszun MC , Alawad AS , Lingala S , et al. , 2020 . Vitamin E treatment in NAFLD patients demonstrates that oxidative stress drives steatosis through upregulation of de-novo lipogenesis . Redox Biol , 37 : 101710 . https://doi.org/10.1016/j.redox.2020.101710 https://doi.org/10.1016/j.redox.2020.101710 Vitamin E treatment in NAFLD patients demonstrates that oxidative stress drives steatosis through upregulation of de-novo lipogenesis Ravisankar S , Agah S , Kim H , et al. , 2019 . Combined cereal and pulse flavonoids show enhanced bioavailability by downregulating phase II metabolism and ABC membrane transporter function in Caco-2 model . Food Chem , 279 : 88 - 97 . https://doi.org/10.1016/j.foodchem.2018.12.006 https://doi.org/10.1016/j.foodchem.2018.12.006 Combined cereal and pulse flavonoids show enhanced bioavailability by downregulating phase II metabolism and ABC membrane transporter function in Caco-2 model Reddy PH , 2006 . Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of Alzheimer’s disease . J Neurochem , 96 ( 1 ): 1 - 13 . https://doi.org/10.1111/j.1471-4159.2005.03530.x https://doi.org/10.1111/j.1471-4159.2005.03530.x Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of Alzheimer’s disease Rocha M , Apostolova N , Diaz-Rua R , et al. , 2020 . Mitochondria and T2D: role of autophagy, ER stress, and inflammasome . Trends Endocrinol Metab , 31 ( 10 ): 725 - 741 . https://doi.org/10.1016/j.tem.2020.03.004 https://doi.org/10.1016/j.tem.2020.03.004 Mitochondria and T2D: role of autophagy, ER stress, and inflammasome Rowland I , Gibson G , Heinken A , et al. , 2018 . Gut microbiota functions: metabolism of nutrients and other food components . Eur J Nutr , 57 ( 1 ): 1 - 24 . https://doi.org/10.1007/s00394-017-1445-8 https://doi.org/10.1007/s00394-017-1445-8 Gut microbiota functions: metabolism of nutrients and other food components Singh RK , Chang HW , Yan D , et al. , 2017 . Influence of diet on the gut microbiome and implications for human health . J Transl Med , 15 ( 1 ): 73 . https://doi.org/10.1186/s12967-017-1175-y https://doi.org/10.1186/s12967-017-1175-y Influence of diet on the gut microbiome and implications for human health Singleton VL , Rossi JA , 1965 . Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents . Am J Enol Vitic , 16 ( 3 ): 144 - 158 . Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents Siracusa L , Kulisic-Bilusic T , Politeo O , et al. , 2011 . Phenolic composition and antioxidant activity of aqueous infusions from Capparis spinosa L . and Crithmum maritimum L. before and after submission to a two-step in vitro digestion model. J Agric Food Chem , 59 ( 23 ): 12453 - 12459 . https://doi.org/10.1021/jf203096q https://doi.org/10.1021/jf203096q Phenolic composition and antioxidant activity of aqueous infusions from Capparis spinosa L Spagnuolo C , Napolitano M , Tedesco I , et al. , 2016 . Neuroprotective role of natural polyphenols . Curr Top Med Chem , 16 ( 17 ): 1943 - 1950 . https://doi.org/10.2174/1568026616666160204122449 https://doi.org/10.2174/1568026616666160204122449 Neuroprotective role of natural polyphenols Su HM , Xie LH , Xu Y , et al. , 2020 . Pelargonidin-3- O -glucoside derived from wild raspberry exerts antihyperglycemic effect by inducing autophagy and modulating gut microbiota . J Agric Food Chem , 68 ( 46 ): 13025 - 13037 . https://doi.org/10.1021/acs.jafc.9b03338 https://doi.org/10.1021/acs.jafc.9b03338 Pelargonidin-3-O-glucoside derived from wild raspberry exerts antihyperglycemic effect by inducing autophagy and modulating gut microbiota Tseng CY , Wang JS , Chao MW , 2017 . Causation by diesel exhaust particles of endothelial dysfunctions in cytotoxicity, pro-inflammation, permeability, and apoptosis induced by ROS generation . Cardiovasc Toxicol , 17 ( 4 ): 384 - 392 . https://doi.org/10.1007/s12012-016-9364-0 https://doi.org/10.1007/s12012-016-9364-0 Causation by diesel exhaust particles of endothelial dysfunctions in cytotoxicity, pro-inflammation, permeability, and apoptosis induced by ROS generation Wang B , 2011 . Chemical characterization and ameliorating effect of polysaccharide from Chinese jujube on intestine oxidative injury by ischemia and reperfusion . Int J Biol Macromol , 48 ( 3 ): 386 - 391 . https://doi.org/10.1016/j.ijbiomac.2010.12.005 https://doi.org/10.1016/j.ijbiomac.2010.12.005 Chemical characterization and ameliorating effect of polysaccharide from Chinese jujube on intestine oxidative injury by ischemia and reperfusion Wang SM , Huang YW , Xu HH , et al. , 2017 . Oxidized tea polyphenols prevent lipid accumulation in liver and visceral white adipose tissue in rats . Eur J Nutr , 56 ( 6 ): 2037 - 2048 . https://doi.org/10.1007/s00394-016-1241-x https://doi.org/10.1007/s00394-016-1241-x Oxidized tea polyphenols prevent lipid accumulation in liver and visceral white adipose tissue in rats Yahfoufi N , Alsadi N , Jambi M , et al. , 2018 . The immunomodulatory and anti-inflammatory role of polyphenols . Nutrients , 10 ( 11 ): 1618 . https://doi.org/10.3390/nu10111618 https://doi.org/10.3390/nu10111618 The immunomodulatory and anti-inflammatory role of polyphenols Zhang L , Liu PZ , Li LL , et al. , 2018 . Identification and antioxidant activity of flavonoids extracted from Xinjiang jujube ( Ziziphus jujube Mill .) leaves with ultra-high pressure extraction technology . Molecules , 24 ( 1 ): 122 . https://doi.org/10.3390/molecules24010122 https://doi.org/10.3390/molecules24010122 Identification and antioxidant activity of flavonoids extracted from Xinjiang jujube (Ziziphus jujube Mill.) leaves with ultra-high pressure extraction technology Zhang WJ , Liu C , Yang RJ , et al. , 2019 . Comparison of volatile profiles and bioactive components of sun-dried Pu-erh tea leaves from ancient tea plants on Bulang Mountain measured by GC-MS and HPLC . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 20 ( 7 ): 563 - 575 . http://doi.org/10.1631/jzus.B1800183 http://doi.org/10.1631/jzus.B1800183 Comparison of volatile profiles and bioactive components of sun-dried Pu-erh tea leaves from ancient tea plants on Bulang Mountain measured by GC-MS and HPLC Zhao HX , Zhang HS , Yang SF , 2014 . Phenolic compounds and its antioxidant activities in ethanolic extracts from seven cultivars of Chinese jujube . Food Sci Hum Well , 3 ( 3-4 ): 183 - 190 . https://doi.org/10.1016/j.fshw.2014.12.005 https://doi.org/10.1016/j.fshw.2014.12.005 Phenolic compounds and its antioxidant activities in ethanolic extracts from seven cultivars of Chinese jujube Zhao XR , Du GC , Zou HJ , et al. , 2013 . Progress in preventing the accumulation of ethyl carbamate in alcoholic beverages . Trends Food Sci Technol , 32 ( 2 ): 97 - 107 . https://doi.org/10.1016/j.tifs.2013.05.009 https://doi.org/10.1016/j.tifs.2013.05.009 Progress in preventing the accumulation of ethyl carbamate in alcoholic beverages Zhu XR , Wang H , Sun J , et al. , 2019 . Pericarp and seed of litchi and longan fruits: constituent, extraction, bioactive activity, and potential utilization . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 20 ( 6 ): 503 - 512 . Pericarp and seed of litchi and longan fruits: constituent, extraction, bioactive activity, and potential utilization Tao BAO performed fermentation experiments, HPLC analysis, in vitro antioxidant activity and cell experiments, and wrote the initial manuscript. Ming ZHANG performed in vitro antioxidant activity experiments and wrote the initial manuscript. Yuanqing ZHOU performed fermentation experiments and HPLC analysis. Wei CHEN conceived the project, designed the study, and supervised the experiments. All authors have read and approved the final manuscript. Therefore, all authors have full access to all the data in the study and take responsibility for its integrity and security. This work was supported by the National Natural Science Foundation of China (Nos. U1703105 and 21876152) and the Lishui Science and Technology Bureau Research Fund (No. 2020zdhz01), China. 10.1631/jzus.B2000754 1673-1581（2021）05-0397-13 2020-11-23 2021-01-18 2021-05-15 2022-05-09 2021-03-30 © Zhejiang University Press 2021 Tao BAO, Ming ZHANG, Yuanqing ZHOU, and Wei CHEN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 22