Nat Prod Sci.  2016 Mar;22(1):53-59. 10.20307/nps.2016.22.1.53.

Dihydrobenzofuran Neolignans Isolated from Euonymus alatus Leaves and Twigs Attenuated Inflammatory Responses in the Activated RAW264.7 Macrophage Cells

• Kim NH
• Yang MH
• Heo JD
• Sung SH
• Jeong EJ

• KMID: 2312922
• DOI: http://doi.org/10.20307/nps.2016.22.1.53

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 pro-inflammatory 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.

Keyword

Euonymus alatus; Neolignan; Anti-inflammation; RAW264.7

MeSH Terms

Cytokines
Dinoprostone
Euonymus*
Interleukin-6
Lignans*
Macrophages*
Necrosis
Nitric Oxide
Nitric Oxide Synthase Type II
Cytokines
Dinoprostone
Interleukin-6
Lignans
Nitric Oxide
Nitric Oxide Synthase Type II

Figure

• 

  Fig. 1. Structures of compounds 1–6 isolated from E. alatus leaves and twigs.

• 

  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.

• 

  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.

• 

  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.

• 

  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.

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