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  • br Statistical analysis br Data are presented as

    2022-05-06


    Statistical analysis
    Data are presented as means ± standard deviations. Student's t-test was used to compare two independent samples. Groups were also compared using a one-way analysis of variance with Tukey's post hoc test for significant main effects (SPSS 12.0 K for Windows; SPSS Inc., Chicago, IL, USA).
    Results
    Comparison of components of enzymatic hydrolysis of citrus unshiu peel (εCUP) and fermented extraction of citrus unshiu peel (ƒCUP)
    Previously, we demonstrated that the products of enzymatic hy-drolysis of Citrus unshiu peel (εCUP) and fermented extraction of Citrus unshiu peel (ƒCUP) induced different anti-cancer effects on human pancreatic cancer both in vitro and in vivo. In addition, we verified that εCUP and ƒCUP consist of narirutin, hesperidin, naringenin, and he-speretin. Of note, newly generated naringenin and hesperetin by en-zymatic hydrolysis or fermentation process were the critical compo-nents of anti-cancer effects (Lee et al., 2017a; Lee et al., 2018). To demonstrate whether the ratio of naringenin and hesperetin can affect the natural features of εCUP and ƒCUP, we performed HPLC analysis using εCUP and ƒCUP separately (Fig. 2A). Then, we calculated the LOD and LOQ values of the four components in εCUP and ƒCUP. The yields of narirutin, hesperidin, naringenin, and hesperetin in εCUP were 110.6 ± 1.04 ppm, 250.63 ± 50.27 ppm, 30.12 ± 1.84 ppm, and 10.67 ± 1.84 ppm, respectively; while the yields of narirutin, hesper-idin, naringenin, and hesperetin in ƒCUP were 35.74 ± 1.04 ppm, 400.86 ± 50.27 ppm, 36.20 ± 1.84 ppm, and 76.22 ± 1.84 ppm, respectively (Fig. 2B). These results suggest that εCUP and ƒCUP have different ratios of naringenin and hesperetin.
    In vitro effect of naringenin and hesperetin combined treatment
    To evaluate the anti-cancer effects of naringenin and hesperetin combined treatment, we firstly treated solely naringenin or hesperetin in Miapaca-2, Panc-1, and SNU-213 Relebactam at various doses (0, 1, 5, 10, and 20 µM) over 3 days. Miapaca-2, Panc-1, and SNU-213 cells showed little sensitivity to naringenin or hesperetin treatment alone (Fig. 3A). Next, we investigated the combined effects on human pancreatic cancer cells of naringenin and hesperetin in accordance with the natural ratio  Phytomedicine 58 (2019) 152762
    in εCUP and ƒCUP. Various combined treatments of narigenin and he-speretin showed different anti-viability effects in Miapaca-2, Panc-1, and SNU-213 cells. The εCUP mimic condition (naringenin:hesper-etin = 75:25) showed the most effective anti-viability effect compared with ƒCUP mimic condition (naringenin:hesperetin = 35:65) and ba-lanced condition (naringenin:hesperetin = 50:50) (Fig. 3B). To de-termine the effect of optimal ratio with naringenin and hesperetin, we performed a combined treatment with a sublethal dose of gemcitabine and various concentrations of naringenin or hesperetin. Combined treatment with gemcitabine and hesperetin or gemcitabine and nar-ingenin had no synergistic anti-cancer effects in human pancreatic cancer cells such as Panc-1 and SNU-213 cells (Supplementary Fig. 1). Since dying cells by treatment of εCUP mimic condition showed typical morphology of apoptotic cell death, we assessed caspase-3-mediated apoptosis in Panc-1 cells. Treatment with combined treatment of nar-ingenin and hesperetin induced caspase-3 cleavage in Panc-1 cells; however, pre-treatment with a pan-caspase inhibitor, z-VAD-FMK, re-sulted in no cleavage of procaspase-3 (Fig. 3C). In addition, combined treatment of narigenin and hesperetin significantly reduced migration of Miapaca-2, Panc-1, and SNU-213 cells at relatively Relebactam low dosages (Fig. 3D). The εCUP mimic condition also showed the most effective anti-migration effect compared with ƒCUP mimic condition and ba-lanced condition.
    To evaluate the basal toxicity of naringenin and hesperetin com-bined treatment in normal cells, we used HUVECs and human normal fibroblast Detroit551 cells. Treatment with the most effective εCUP mimic condition (narigenin: 7.5 µM, hesperetin: 2.5 µM) significantly inhibited cell viability of Panc-1 cell. However, it had no anti-viability effect on HUVEC and Detroit551 (Fig. 4A). Treatment with εCUP mimic condition also significantly inhibited Panc-1 cell migration, but it had no inhibitory effect on HUVEC and Detroit551 (Fig. 4B). We further assessed the toxicity of gemcitabine, a conventional chemotherapeutic reagent in HUVECs and Detroit551 cells to compare with εCUP mimic condition. Gemcitabine significantly induced an anti-viability effect on Panc-1 cells even at the lower concentration than εCUP mimic condi-tion. However, gemcitabine also significantly reduced HUVECs and Detroit551 viability at the same dosages (Fig. 4C). These results suggest that combined treatment of naringenin and hesperetin effectively in-duces anti-cancer effects in human pancreatic cancer cells without af-fecting normal cells.