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Protective Effects of Sweet Orange, Unshiu Mikan, and Mini Tomato Juice Powders on t-BHP-Induced Oxidative Stress in HepG2 Cells - PubMed

Protective Effects of Sweet Orange, Unshiu Mikan, and Mini Tomato Juice Powders on t-BHP-Induced Oxidative Stress in HepG2 Cells

Susoma Jannat et al. Prev Nutr Food Sci. 2016 Sep.

Abstract

The aim of this study was to investigate the protective effects of juice powders from sweet orange [Citrus sinensis (L.) Osbeck], unshiu mikan (Citrus unshiu Marcow), and mini tomato (Solanum lycopersicum L.), and their major flavonoids, hesperidin, narirutin, and rutin in tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in HepG2 cells. The increased reactive oxygen species and decreased glutathione levels observed in t-BHP-treated HepG2 cells were ameliorated by pretreatment with juice powders, indicating that the hepatoprotective effects of juice powders and their major flavonoids are mediated by induction of cellular defense against oxidative stress. Moreover, pretreatment with juice powders up-regulated phase-II genes such as heme oxygenase-1 (HO-1), thereby preventing cellular damage and the resultant increase in HO-1 expression. The high-performance liquid chromatography profiles of the juice powders confirmed that hesperidin, narirutin, and rutin were the key flavonoids present. Our results suggest that these fruit juice powders and their major flavonoids provide a significant cytoprotective effect against oxidative stress, which is most likely due to the flavonoid-related bioactive compounds present, leading to the normal redox status of cells. Therefore, these fruit juice powders could be advantageous as bioactive sources for the prevention of oxidative injury in hepatoma cells.

Keywords: flavonoids; fruit juice powder; heme oxygenase-1; hepatoprotection; t-BHP.

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Figures

Fig. 1
Fig. 1

HPLC chromatograms of standard (A), unshiu mikan (B), sweet orange (C), and boiled tomato (D). 1, hesperidin; 2, narirutin; 3, rutin (E).

Fig. 2
Fig. 2

Effects of unshiu mikan (A), sweet orange (B), boiled tomato (C), raw tomato (D), hesperidin (E), rutin (F), and narirutin (G) on cell viability in HepG2 cells. Cells were pretreated with the indicated concentrations of 50~200 μg/mL of juice powders from oranges and boiled tomatoes and with hesperidin, rutin, and narirutin at concentrations of 2.5 to 100 μM for 24 h. Data shown represent mean±standard deviation of triplicate experiments.

Fig. 3
Fig. 3

Cytoprotective effects of unshiu mikan (A), sweet orange (B), boiled tomato (C), hesperidin (D), rutin (E), and narirutin (F) on tert-butyl hydroperoxide (t-BHP)-treated HepG2 cells. Cells were pretreated with the indicated concentrations (50 to 200 μg/mL) of juice powders and with hesperidin, rutin, and narirutin at concentrations of 2.5 to 100 μM for 24 h. After that time, cells were treated with t-BHP (200 μM) and incubated for 2 h. Control values were obtained in the absence of t-BHP and juice powders and flavonoids. Silymarin was used as a positive control. Data are presented as mean±SD of triplicate experiments. Significantly different from the t-BHP-treated group at *P<0.05 and the control group at ### P<0.001.

Fig. 4
Fig. 4

Effects of unshiu mikan (A), sweet orange (B), boiled tomato (C), hesperidin (D), rutin (E), and narirutin (F) on tert-butyl hydroperoxide (t-BHP)-induced reactive oxygen species (ROS) generation in HepG2 cells. Cells pretreated with different concentrations (50 to 200 μg/mL) of juice powders and hesperidin, rutin, and narirutin at concentrations of 2.5 to 100 μM or Trolox (10 μM) for 1 h were stimulated with 200 μM t-BHP for 30 min. ROS levels were measured by 2′,7′-dichlorodihydrofluorescein diacetate with fluorescent analysis. The control values were obtained in the absence of t-BHP (200 μM), juice powders, and flavonoids and after the addition of t-BHP (200 μM). Trolox was used as a positive control. Data are expressed as the mean±SD of three independent experiments. Significantly different from the t-BHP-treated group at *P<0.05 and the control group at ###P<0.001.

Fig. 5
Fig. 5

Effects of unshiu mikan (A), sweet orange (B), boiled tomato (C), hesperidin (D), rutin (E), and narirutin (F) on intracellular glutathione (GSH) level in tert-butyl hydroperoxide (t-BHP)-treated HepG2 cells. Cells were pretreated with the indicated concentrations (50 to 200 μg/mL) of juice powders and with hesperidin, rutin, and narirutin at concentrations of 2.5 to 100 μM for 24 h. Then t-BHP (200 μM) was added, and cells were incubated for 2 h. Whole cell proteins were isolated and used for GSH assays. The data shown represent the mean±SD of triplicate experiments. Significantly different from the t-BHP-treated group at *P<0.05 and the control group at ### P<0.001.

Fig. 6
Fig. 6

Effects of unshiu mikan (A), sweet orange (B), boiled tomato (C), hesperidin (D), rutin (E), and narirutin (F) on heme oxygenase-1 (HO-1) protein expression. Cells were exposed to various concentrations of juice powders and hesperidin, rutin, and narirutin for 24 h, and expression levels of proteins, including HO-1, were analyzed by Western blotting.

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