Journal List > Nat Prod Sci > v.22(1) > 1060641

Kim, Yang, Heo, Sung, and Jeong: Dihydrobenzofuran Neolignans Isolated from Euonymus alatus Leaves and Twigs Attenuated Inflammatory Responses in the Activated RAW264.7 Macrophage Cells

Abstract

Anti-inflammatory effects of dihydrobenzofuran neolignans isolated from Euonymus alatus leaves and twigs were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Six neolignans, (+)-simulanol (1), (+)-dehydrodiconiferyl alcohol (2), (−)-simulanol (3), (−)-dehydrodiconiferyl alcohol (4), (+)-dihydrodehyrodiconiferyl alcohol (5), threo-buddlenol B (6) effectively inhibited the production of nitric oxide (NO) induced by LPS, and the activity of iNOS. (−)-dehydrodiconiferyl alcohol (4), which showed the most potent inhibitory activity, attenuated the activity of iNOS enzyme and also the expression of iNOS and COX-2 proteins. The subsequent production of proinflammatory cytokines, interleukin-1β, interleukin-6, tumor necrosis factor-α and prostaglandin E2 were also inhibited by the pretreatment of RAW264.7 cells with (−)-dehydrodiconiferyl alcohol (4). These neolignans are thought to contribute to anti-inflammatory effects of E. alatus, and expected to be potential candidates to prevent/treat inflammation-related diseases.

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Fig. 1.
Structures of compounds 1–6 isolated from E. alatus leaves and twigs.
nps-22-53f1.tif
Fig. 2.
Inhibitory effects of compounds 1–6 on iNOS enzyme activity in LPS-stimulated RAW264.7 cells. RAW264.7 cells were treated with compounds 1–6 for 1 h before exposure to LPS for 24 h. The fluorescent product was assessed as described in the Materials and Methods. The values shown are the mean ± s.d. of data from three independent experiments. Results differ significantly from LPS alone,P < 0.01,∗∗P < 0.001.
nps-22-53f2.tif
Fig. 3.
Inhibitory effects of compounds 4 on the expression of iNOS protein in LPS-stimulated RAW264.7 cells. RAW264.7 cells were treated with compound 4 for 1 h, and then exposed to LPS for 24 h. Cell lysates (40 ug protein) were prepared and subjected to Western blot analysis using iNOS-specific antibodies. The relative protein levels were quantified by scanning densi-tometry and normalized to b-tubulin protein. NO production was measured by the Griess reaction and sodium nitrite was used as a standard. The values shown are the mean ± s.d. of data from three independent experiments. Results differ significantly from LPS alone,P < 0.01,∗∗P < 0.001.
nps-22-53f3.tif
Fig. 4.
Inhibitory effects of compounds 4 on the expression of COX-2 protein in LPS-stimulated RAW264.7 cells. RAW264.7 cells were treated with compound 4 for 1 h, and then exposed to LPS for 24 h. Cell lysates (40 ug protein) were prepared and subjected to Western blot analysis using COX-2-specific antibodies. The relative protein levels were quantified by scanning densito-metry and normalized to b-tubulin protein. The concentrations of PGE2 in the culture medium were determined using ELISA system as described in Materials and Methods. The values shown are the mean ± s.d. of data from three independent experiments. Results differ significantly from LPS alone,P <0.01,∗∗P <0.001.
nps-22-53f4.tif
Fig. 5.
Inhibitory effects of compounds 4 on the production of proinflammatory cytokines in LPS-stimulated RAW264.7 cells. RAW264.7 cells were treated with compound 4 for 1 h, and then exposed to LPS for 24 h. The concentrations of IL-1β, IL-6 and TNF-α in the culture medium were determined using ELISA system as described in Materials and Methods. The values shown are the mean ± s.d. of data from three independent experiments. Results differ significantly from LPS alone,P <0.01,∗∗P <0.001.
nps-22-53f5.tif
Table 1.
Inhibitory effects of compounds 1–6 isolated from E. alatus leaves and twigs on NO production induced by LPS in RAW264.7 cells
Concentration 10 μ M 20 μ M 50 μ M 100 μ M IC50 μ M
Nitrite (μ M)
Control 5.1 ± ± 0.2
LPS 56.5 ±0.3
1 33.2 ± 1.9 20.2 ± 1.4 18.8 ± 2.7 15.4 ± 0.8∗∗ 11.7 ± 1.2
2 29.3 ± 2.3 23.2 ± 1.6 11.2 ± 1.2∗∗ 66.1 ± 0.1∗∗ 9.3 ± 1.4
3 32.9 ± 1.7 26.4 ± 0.3 17.3 ± 1.5∗∗ 14.9 ± 0.5∗∗ 12.8 ± 2.0
4 26.6 ± 2.0 21.4 ± 1.5 68.7 ± 0.2∗∗ 62.9 ± 0.1∗∗ 8.5 ± 0.8
5 31.7 ± 1.5 24.2 ± 1.2 12.7 ± 0.8∗∗ 66.9 ± 0.2∗∗ 9.8 ± 2.0
6 48.8 ± 2.5 31.9 ± 0.9 29.8 ± 1.3 28.9 ± 1.0 21.3 ± 3.3
L-NAME         48.5 ± 2.3
L-NNA         60.3 ± 1.7
L-NMMA         32.9 ± 2.2

RAW 264.7 cells were pre-treated with each compound for 1 h before exposure to LPS for 24 h. The concentration of nitrite in culture medium was measured as described in the Methods. The values shown are mean ± s.d. of data from three independent experiments. Significant compared with LPS alone

P < 0.001,

∗∗ P < 0.0001. L-NAME (L-Nitro-Arginine Methyl Ester, the NOS inhibitor, L-NNA (NG-Nitro-L-arginine, a well known inhibitor of NOS) and L-NMMA (NG-methyl-L-arginine acetate, an inhibitor of iNOS-induced nitric oxide production) were used as positive controls.

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