Anti-inflammatory effects of Viola yedoensis and the application of cell extraction methods for investigating bioactive constituents in macrophages

Abstract

Background: Viola yedoensis (VY, Violaceae) is a popular medicinal herb used in traditional eastern medicine for treating lots of diseases, including inflammation and its related symptoms. However, the anti-inflammatory properties of VY have not been demonstrated. In the present study, we investigated the anti-inflammatory effects of VY ethanol extract (VYE) on macrophages and attempted to identify the bioactive components of VYE.

Methods: We assessed the effects of VYE on secretion of nitric oxide (NO) and inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. In addition, we explored the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and changes in heme oxygenase (HO)-1, nuclear factor (NF)-kB, and mitogen-activated protein kinase (MAPK) signaling pathways in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). In addition, a rapid and useful approach to identify potential bioactive components in VYE with anti-inflammatory effects was developed using murine macrophage cell extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS).

Results: We found that VYE exerted anti-inflammatory activity by inhibiting the production of key inflammation mediators and related products, as well as suppression of HO-1, NF-kB, and MAPK signaling pathway activation in RAW 264.7 cells. In addition, we identified two compounds in VYE via the cell extraction method.

Conclusions: Our results revealed that VYE exerts anti-inflammatory activities and its detailed inhibitory mechanism in macrophages. Furthermore, we identified bioactive components of VYE.

Keywords: Bio-active components; Cell extraction; Heme oxygenase-1; Mitogen-activated protein kinase; Nuclear factor-kappaB; Viola yedoensis ethanol extract.

Fig. 1

a Cytotoxicity of VYE in RAW 264.7 macrophages and the suppressive effect of VYE on (b) NO secretion and (c–e) TNF-α, IL-6, and IL-1β cytokine production upon LPS stimulation. RAW 264.7 cells were pretreated with VYE for 1 h prior to incubation with LPS for 24 h. Cytotoxicity was determined by CCK assay. NO content in the conditioned medium was determined using Griess reagent. TNF-α, IL-6, and IL-1β levels in the medium, as measured by ELISA. As a control, cells were incubated with vehicle alone. Data represent the means ± SE of three independent experiments. ** p < 0.001 compared to the LPS-stimulated value

Fig. 2

Effect of VYE on LPS-induced (a) TNF-α, (b) IL-6, and (c) IL-1β mRNA expression in macrophages. Cells were pretreated with VYE for 1 h and stimulated with LPS for a further 6 h. mRNA levels were analyzed by real-time RT-PCR. Amplification and analyses of mRNA were performed using the QuantStudio 6 Flex Real-time PCR System (Thermo Scientific, Rockford, IL, USA). Data represent the means ± SE of three independent experiments. * p < 0.01 and ** p < 0.001 compared to the LPS-stimulated value

Fig. 3

Effect of VYE on (a) iNOS, COX-2 protein levels, and (b) HO-1 protein induction in RAW 264.7 cells. Cells were treated with (a) LPS alone or LPS plus VYE for 24 h or with (b) VYE alone for 6 h. Protein levels were determined by Western blotting, as described in the Materials and Methods, and quantitated using a Davinch-chemi™ CAS-400SM chemiluminescence imaging system (Core Bio, Seoul, Korea). β-actin served as a control. The experiment was repeated three times independently, and similar results were obtained

Fig. 4

Effects of VYE on mRNA expression of (a) iNOS, (b) COX-2, and (c) HO-1 in macrophages. Cells were treated with (a, b) LPS alone or with LPS and VYE for 24 h and (c) VYE alone for 3 h. Data represent the means ± SE of determinations from three independent experiments. ** p < 0.001 compared to the LPS-stimulated value

Fig. 5

Effects of VYE on LPS-induced (a) translocation of NF-kB p65 into the nucleus and (b) phosphorylation of IkBα in RAW 264.7 cells. Cells were pretreated with VYE for 1 h and stimulated with LPS for a further (a) 1 h or (b) 30 min. β-actin and TATA box-binding protein (TBP) were used as control proteins of the cytosolic and nuclear fractions, respectively. The experiment was repeated three times independently, and similar results were obtained

Fig. 6

Effects of VYE on the phosphorylation of (a) ERK, (b) p38, and (c) JNK MAPKs in LPS-stimulated macrophages. Cells were treated with VYE for 1 h and challenged with LPS for 30 min. Proteins were examined by Western blot analysis. ERK, p38 and JNK were used as controls for their phosphorylated forms. The experiment was repeated three times independently, and similar results were obtained

Fig. 7

Effect of VYE on LPS-induced (a) TNF-α, (b) IL-6, and (c) IL-1β cytokine production in mouse peritoneal macrophages. Mouse peritoneal macrophage cells were treated with VYE for 1 h prior to incubation with LPS for 24 h. Cytokine levels in the culture supernatant were analyzed by ELISA. As a control, the cells were incubated with vehicle alone. Data represent the means ± SE of three independent experiments. ** p < 0.001 compared to the LPS-stimulated value

Fig. 8

UV chromatogram (330 nm). a Desorption eluate of RAW 264.7 cells incubated without VYE, b fourth wash eluate of cell extract, c desorption eluate of cells incubated with VYE, and d extract of VYE

Fig. 9

Mass chromatogram (negative mode). a Desorption eluate of RAW 264.7 cells incubated without VYE, b fourth wash eluate of cell extract, c desorption eluate of cells incubated with VYE, and d extract of VYE

Fig. 10

(A) Extracted ion chromatogram (EIC) of VYE extract for 563 and (B) extracted ion chromatogram (EIC) of VYE extract for 533. (A) Desorption eluate of RAW 264.7 cells incubated without VYE, (B) fourth wash eluate of cell extract, (C) desorption eluate of cells incubated with VYE, and (D) extract of VYE

Fig. 11

Isolation from V. yedoensis

Fig. 12

¹H- and ¹³C-NMR spectra of compound 1

Fig. 13

¹H- and ¹³C-NMR spectra of compound 2