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. 2022 Dec 5;27(23):8568.
doi: 10.3390/molecules27238568.

Geniposidic Acid from Eucommia ulmoides Oliver Staminate Flower Tea Mitigates Cellular Oxidative Stress via Activating AKT/NRF2 Signaling

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Geniposidic Acid from Eucommia ulmoides Oliver Staminate Flower Tea Mitigates Cellular Oxidative Stress via Activating AKT/NRF2 Signaling

Shuo Cheng et al. Molecules. .

Abstract

Eucommia ulmoides Oliver staminate flower (ESF) tea enjoys a good reputation in folk medicine and displays multiple bioactivities, such as antioxidant and antifatigue properties. However, the underlying biological mechanisms remain largely unknown. In this study, we aimed to investigate whether ESF tea can mitigate cellular oxidative stress. Crude ethyl alcohol extract and its three subfractions prepared by sequential extraction with chloroform, n-butyl alcohol and residual water were prepared from ESF tea. The results of antioxidant activity tests in vitro manifested n-butyl alcohol fraction (n-BUF) showed the strongest antioxidant capacity (DPPH: IC50 = 24.45 ± 0.74 μg/mL, ABTS: IC50 = 17.25 ± 0.04 μg/mL). Moreover, all subfractions of ESF tea, especially the n-BUF, exhibited an obvious capacity to scavenge the reactive oxygen species (ROS) and stimulate the NRF2 antioxidative response in human keratinocytes HaCaT treated by H2O2. Using ultra-high-performance liquid chromatography, we identified geniposidic acid (GPA) as the most abundant component in ESF tea extract. Furthermore, it was found that GPA relieved oxidative stress in H2O2-induced HaCaT cells by activating the Akt/Nrf2/OGG1 pathway. Our findings indicated that ESF tea may be a source of natural antioxidants to protect against skin cell oxidative damage and deserves further development and utilization.

Keywords: AKT; Eucommia ulmoides Oliver staminate flower tea; NRF2; geniposidic acid; keratinocyte; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ESF tea ameliorated cytotoxicity and attenuated intracellular ROS generation in H2O2-treated HaCaT cells. (A) HaCaT cells were treated with four extract fractions at the indicated concentrations (10, 20, 50, 100 μg/mL) for 24 h. Cell viability was evaluated by a CCK−8 kit. (B) The effect of H2O2 on human immortalized keratinocyte (HaCaT) cell viability was evaluated by CCK−8. (C) HaCaT cells were treated with four extract fractions for 24 h before being exposed to H2O2 (750 μM). The protective effect of tea extract on cell viability in H2O2-treated cells was evaluated by CCK−8 kit. (D) HaCaT were cultured in 35 mm dishes, treated with different extract fractions (50 μg/mL) for 24 h and then exposed to 750 μM H2O2. Intracellular ROS generation was measured by a fluorescence microscope using CM−H2DCFDA probe. (E) Under the same experimental conditions, intracellular ROS intensity was measured with a SpectraMax i3X microplate reader (Molecular Devices, CA, USA) and compared to the control group. Data were represented as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
ESF tea protects HaCaT against H2O2−induced oxidative injury via modulating antioxidative protein expression. Cells were exposed to H2O2 (750 μM) after being pretreated with four ESF tea extract fractions (50 μg/mL) for 24 h. (A) Western blotting was performed to identify the expression level of HO-1, NQO1, GCLM and NRF2. β-actin was used as the internal control. (B) The quantitative analysis of the band intensity by ImageJ. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
Figure 3
Figure 3
UHPLC chromatograms of four fractions from ESF tea. UV detection at 240 nm. Peak name: 1: geniposidic acid, 2: chlorogenic acid, 3: catechin, 4: caffeic acid, 5: geniposide, 6: rutin.
Figure 4
Figure 4
GPA reduced oxidative stress induced by H2O2 through NRF2/OGG1 signaling. (A) HaCaT cells were treated with GPA (50 μM) for 24 h before being exposed to H2O2 with indicated concentrations (250 μM, 500 μM, 750 μM) for 4 h. Clonogenic surviving assay was used to evaluate the effects of GPA pretreatment on clonogenicity under oxidative stress. (B) Cells were exposed to H2O2 (750 μM) for 4 h after being pretreated with GPA (50 μM) for 24 h. Intracellular ROS generation was measured by a fluorescence microscope using a CM−H2DCFDA probe. (C) In a parallel experiment, the DCF fluorescence intensity was measured with a SpectraMax i3X microplate reader (Molecular Devices, CA, USA) and compared to the control group. (D) Cells were exposed to H2O2 (750 μM) for 4 h after being pretreated with GPA (10 μM, 25 μM, 50 μM) for 24 h. Western blotting was performed to identify the antioxidative genes expression level. (E) The cells were pretreated with GPA (50 μM) for 12 h and then cotreated with ML385 (10 μM) for 12 h before being exposed to H2O2 (750 μM). After 4 h, the 8-OHdG level of cells was measured using immunofluorescent staining. (F) Under the same experimental conditions, the expression levels of NRF2 and OGG1 were determined by Western blotting analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
Figure 4
Figure 4
GPA reduced oxidative stress induced by H2O2 through NRF2/OGG1 signaling. (A) HaCaT cells were treated with GPA (50 μM) for 24 h before being exposed to H2O2 with indicated concentrations (250 μM, 500 μM, 750 μM) for 4 h. Clonogenic surviving assay was used to evaluate the effects of GPA pretreatment on clonogenicity under oxidative stress. (B) Cells were exposed to H2O2 (750 μM) for 4 h after being pretreated with GPA (50 μM) for 24 h. Intracellular ROS generation was measured by a fluorescence microscope using a CM−H2DCFDA probe. (C) In a parallel experiment, the DCF fluorescence intensity was measured with a SpectraMax i3X microplate reader (Molecular Devices, CA, USA) and compared to the control group. (D) Cells were exposed to H2O2 (750 μM) for 4 h after being pretreated with GPA (10 μM, 25 μM, 50 μM) for 24 h. Western blotting was performed to identify the antioxidative genes expression level. (E) The cells were pretreated with GPA (50 μM) for 12 h and then cotreated with ML385 (10 μM) for 12 h before being exposed to H2O2 (750 μM). After 4 h, the 8-OHdG level of cells was measured using immunofluorescent staining. (F) Under the same experimental conditions, the expression levels of NRF2 and OGG1 were determined by Western blotting analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
Figure 5
Figure 5
Effects of GPA and inhibition of AKT on phosphorylation of AKT and expression level of NRF2 and its downstream genes. (A) Cells were co-pretreated with GPA (50 μM) and MK2206 (10 μM) for 24 h and then exposed to 750 μM H2O2 for 4 h. Western blotting was performed to identify the corresponding genes’ expression levels. (B) HaCaT cells were pretreated with GPA (50 μM), and SC66 (5 μM) for 24 h, followed by treatment with H2O2 (750 μM) for another 4 h. Cell lysates were collected and then used for Western blotting analysis. (C) Under the same experimental conditions, intracellular ROS generation was measured by a fluorescence microscope using a CM−H2DCFDA probe. (D) In a parallel experiment, the DCF fluorescence intensity was measured with a SpectraMax i3X microplate reader (Molecular Devices, CA, USA) and compared to the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
The schematic figure of this study. CE and its three subfractions were prepared by sequential extraction from ESF tea. The active components and antioxidant function were compared among four fractions. As the main active component in ESF tea extract, GPA protected cells against oxidative stress via the AKT/NRF2/OGG1 pathway.

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