The Flavonoids and Monoterpenes from Citrus unshiu Peel Contained in Ninjinyoeito Synergistically Activate Orexin 1 Receptor: A Possible Mechanism of the Orexigenic Effects of Ninjinyoeito

Abstract

Cancer cachexia, often observed in patients with advanced-stage cancer, is characterized by the loss of body weight and appetite. The Japanese herbal medicine Ninjinyoeito (NYT), which is composed of 12 crude herbal components, has been used as a therapeutic in Japan to improve anorexia and fatigue, which are commonly observed in cancer patients with cachexia. We have previously reported that Citrus unshiu peel (CUP) contained in NYT can enhance food intake by activating the orexin 1 receptor (OX1R). Using the CellKey™ system, which offers detection of OXR activity in intracellular impedance changes, NYT and CUP were found to activate OX1R, which in turn was inhibited by SB-674042, a selective OX1R antagonist. Among the flavonoids contained in CUP, nobiletin and hesperidin, but not naringin, activated OX1R. Furthermore, some monoterpenes contained in CUP, including limonene and linalool, but not terpineol, activated OX1R. In addition, nobiletin and limonene synergistically activated OX1R when added simultaneously. However, neither NYT nor CUP induced OX2R activity. The results collectively suggested that the CUP contained in NYT activates OX1R, but not OX2R, and that flavonoids and monoterpenes in CUP can synergistically activate OX1R. These findings could provide evidence supporting the therapeutic potential of NYT in cancer patients with cachexia.

Keywords: Citrus unshiu peel; Ninjinyoeito; flavonoid; monoterpene; orexin 1 receptor.

Figure 1

Effects of Ninjinyoeito (NYT) and Citrus unshiu peel (CUP) on impedance changes in cells expressing orexin type 1 receptors (OX1R) ((A), n = 9–12), and inhibition of CUP-induced OX1R activities by SB-674042 (SB) ((B), n = 9–26). Data are represented as mean ± SEM. (A) * p < 0.05 vs. vehicle, **** p < 0.0001 vs. vehicle (B) ^†† p < 0.01 vs. veh;cle, ^‡ p < 0.05 vs. CUP alone, Bonferroni’s multiple comparison test. The gray column shows the vehicle, the yellow column shows CUP and the brown one shows NYT. Different orange colors show CUP with different concentrations of SB-674042.

Figure 2

Effects of several flavonoids contained in Citrus unshiu peel (nobiletin, hesperidin, and naringin) on impedance changes in cells expressing OX1R (n = 7). Data are presented as mean ± SEM; (A) ** p < 0.01 vs. vehicle; **** p < 0.0001 vs. vehicle (B) * p < 0.05 vs. vehicle; n.s., not significant (C) n.s., not significant. One-way ANOVA followed by Bonferroni’s multiple comparisons test. The gray column shows the vehicle, the magenta column shows nobiletin, the yellow shows hesperidin, and the pink shows naringin.

Figure 3

Effects of several monoterpenes contained in Citrus unshiu peel [limonene (A), linalool (B), and terpineol (C)] on impedance changes in cells expressing OX1R (n = 9–26). The data are represented as mean ± SEM; (A) * p < 0.05 vs. vehicle; **** p < 0.0001 vs. vehicle (B) **** p < 0.0001 vs. vehicle. Bonferroni’s multiple comparison test. The gray column shows the vehicle, the blue column shows limonene, the green shows linalool, and the dark red shows terpineol.

Figure 4

Synergic effects of limonene and nobiletin in Citrus unshiu peel on impedance changes in cells expressing OX1R (n = 7). Data are presented as mean ± SEM; ^†††† p < 0.0001 vs. vehicle. **** p < 0.0001 vs. limonene alone; n.s., not significant. One-way ANOVA followed by Bonferroni’s multiple comparisons test. The gray column shows the vehicle, the dark gray shows SB-674042 alone, the blue shows limonene, the red shows nobiletin, the purple shows limonene + nobiletin, and the dark purple shows limonene + nobiletin with different concentrations of SB-674042.

Figure 5

Effects of orexin A (A) Ninjinyoeito (NYT), and (C) Citrus unshiu peel (CUP) on (E) impedance changes in cells expressing orexin type 2 receptors (OX2R); and inhibition of orexin A- (B), NYT- (D), and CUP-induced OX2R activities by TCS-OX2-29 (E). Data are presented as mean ± SEM (n = 9 each); (B) ^†††† p < 0.0001 vs. vehicle; **** p < 0.0001 vs. orexin A alone (C) ^†††† p < 0.0001 vs. vehicle (D) ^† p < 0.05 vs. vehicle, n.s., not significant. (E) n.s., not significant. One-way ANOVA followed by Bonferroni’s multiple comparisons test. The gray columns show the vehicle. The blue columns show the results by orexin A, the brown columns show the results by NYT, and the orange columns show the results by CUP.