Ethyl Acetate Fractions of Papaver rhoeas L. and Papaver nudicaule L. Exert Antioxidant and Anti-Inflammatory Activities

Abstract

Abnormal inflammation and oxidative stress are involved in various diseases. Papaver rhoeas L. possesses various pharmacological activities, and a previously reported analysis of the anti-inflammatory effect of P. nudicaule ethanol extracts and alkaloid profiles of the plants suggest isoquinoline alkaloids as potential pharmacologically active compounds. Here, we investigated anti-inflammatory and antioxidant activities of ethyl acetate (EtOAc) fractions of P. nudicaule and P. rhoeas extracts in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. EtOAc fractions of P. nudicaule and P. rhoeas compared to their ethanol extracts showed less toxicity but more inhibitory activity against LPS-induced nitric oxide production. Moreover, EtOAc fractions lowered the LPS-induced production of proinflammatory molecules and cytokines and inhibited LPS-activated STAT3 and NF-κB, and additionally showed significant free radical scavenging activity and decreased LPS-induced reactive oxygen species and oxidized glutathione. EtOAc fractions of P. nudicaule increased the expression of HO-1, GCLC, NQO-1, and Nrf2 in LPS-stimulated cells and that of P. rhoeas enhanced NQO-1. Furthermore, metabolomic and biochemometric analyses of ethanol extracts and EtOAc fractions indicated that EtOAc fractions of P. nudicaule and P. rhoeas have potent anti-inflammatory and antioxidant activities, further suggesting that alkaloids in EtOAc fractions are potent active molecules of tested plants.

Keywords: NF-κB; Nrf2; Papaver nudicaule; Papaver rhoeas; STAT3; anti-inflammatory; antioxidant.

Figure 1

Effects of P. nudicaule and P. rhoeas extracts and ethyl acetate (EtOAc) fraction on cytotoxicity and lipopolysaccharides (LPS)-induced nitric oxide (NO) production. (a) RAW264.7 cells were treated with each ethanol (EtOH) extract and EtOAc fraction of P. nudicaule and P. rhoeas (1.5625–50 μg/mL) for 24 h. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)- and 5-diphenyl-tetrazolium bromide (MTT) assays. (b) RAW264.7 cells were treated with each EtOH extract and the EtOAc fraction of P. nudicaule and P. rhoeas as indicated, and LPS (5 ng/mL) for 24 h. NO production was examined in the culture supernatants. Data are presented as mean ± standard error of the mean (SEM) from at least three independent experiments in triplicate. * p < 0.05, ** p < 0.01 and *** p < 0.001 imply statistically significant differences compared with the LPS only treated group.

Figure 2

Effects of Papaver nudicaule orange (NO-Fr), Papaver nudicaule scarlet (NS-Fr) and Papaver rhoeas red (RA-Fr) on the expression of PGE2, iNOS and COX2. RAW264.7 cells were treated with NO-Fr, NS-Fr and RA-Fr (12.5 and 25 μg/mL), and lipopolysaccharides (LPS) (5 ng/mL) for 24 h. (a) The PGE2 secretion was analyzed, and data are shown as mean ± standard error of the mean (SEM). (b) The expression of iNOS, COX2 and β-actin in the treated cells was investigated by western blotting. (c,d) Quantification of iNOS, COX2 and β-actin expression was performed using the ImageJ software. Expression levels of iNOS and COX2 were normalized to the β-actin protein levels. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01 and *** p < 0.001 imply statistically significant differences compared with the LPS only treated group.

Figure 3

Effects of ethyl acetate (EtOAc) fraction of Papaver rhoeas red (RA-Fr), Papaver nudicaule orange (NO-Fr), Papaver nudicaule scarlet (NS-Fr) on the expression of TNFα, IL-1β and IL-5. RAW264.7 cells were treated with each EtOAc fraction (12.5 and 25 μg/mL) and LPS (5 ng/mL) for 4 h. The expression level of each mRNA was analyzed by normalization to the GAPDH levels. Data are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 and *** p < 0.001 imply statistically significant differences compared with the LPS only treated group.

Figure 4

Effects of ethyl acetate (EtOAc) fraction of Papaver nudicaule orange (NO-Fr), Papaver nudicaule scarlet (NS-Fr), and Papaver rhoeas red (RA-Fr) on the STAT3 and NF-κB signaling pathways. (a,b) Expression and phosphorylation of STAT3 and NF-κB were examined by western blotting in RAW264.7 cells treated as indicated. (c) Expression levels of p-STAT3 were normalized to the STAT3 protein levels. (d) Expression levels of p-IκBα and p-p65 were normalized to the IκBα and p65 protein levels, respectively. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01 and *** p < 0.001 imply statistically significant differences compared with the LPS only treated group.

Figure 5

Effects of ethyl acetate (EtOAc) fraction of Papaver nudicaule orange (NO-Fr), Papaver nudicaule scarlet (NS-Fr), and Papaver rhoeas red (RA-Fr) on free radicals, intracellular reactive oxygen species (ROS) and Glutathione/glutathione disulfide (GSH/GSSG) ratio. (a) 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test by treatment with various concentrations of NO-Fr, NS-Fr and RA-Fr (μg/mL) and ascorbic acid (10 μM) as a positive control. * p < 0.05, ** p < 0.01 and *** p < 0.001 imply statistically significant differences compared with the control group. (b) Cells were treated with NO-Fr, NS-Fr and RA-Fr at two concentrations (12.5 and 25 μg/mL). After treatment for 18 h, ROS generation was measured by DCFH-DA staining with flow cytometry analysis. A graph as a percentage based on lipopolysaccharides (LPS) using mean value. (c) Cells were treated with NO-Fr, NS-Fr and RA-Fr at two concentrations (12.5 and 25 μg/mL). After treatment for 12 h, and the luminescence was measured. The GSH/GSSG ratio is calculated as follows: ratio GSH/GSSG treated = (µM total glutathione treated − (µM GSSG treated × 2))/µM GSSG treated. * p < 0.05, ** p < 0.01 and *** p < 0.001 imply statistically significant differences compared with the LPS only treated group.

Figure 6

Effect of ethyl acetate (EtOAc) fraction of Papaver nudicaule orange (NO-Fr), Papaver nudicaule scarlet (NS-Fr), and Papaver rhoeas red (RA-Fr) on antioxidant genes (HO-1, NQO1 and GCLC) and Nrf2 signaling. RAW264.7 cells were treated with NO-Fr, NS-Fr and RA-Fr (12.5 and 25 μg/mL) and lipopolysaccharides (LPS) (10 ng/mL) for 8 h. (a) Expression of HO-1, NQO1 and GCLC mRNA was analyzed by normalization to the GAPDH mRNA levels. Data are shown as mean ± standard error of the mean (SEM) from at least three independent experiments in triplicate. (b) Nuclear translocation of Nrf2 was examined in RAW264.7 cells treated as indicated. (c) Expression levels of Nuclear Nrf2 shown in (b) were normalized to the Lamin B protein levels. Data are shown as mean ± standard deviation (SD) from at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 imply statistically significant differences compared with the LPS only treated group.

Figure 7

A heat map of bioactive correlation of identified metabolites from ethanol (EtOH) extracts and ethyl acetate (EtOAc) fractions of P. nudicaule and P. rhoeas. Each value in the heat map is a colored representation of a calculated Pearson correlation coefficient of metabolites with NO, ROS, and PGE2. Red and blue colors indicate an increase and decrease of Pearson correlation values, respectively (a). The box plots show the differential peak area of significant seven metabolites analyzed using biochemometrics (b). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001; EtOAc fraction of P. rhoeas (EtOAc-RA) vs. EtOAc fractions of P. nudicaule (EtOAc-N series), EtOH extract of P. rhoeas (EtOH-RA) vs. EtOH extract of P. nudicaule (EtOH-N series), and EtOH-N series vs. EtOAc-N series.