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

Na-Hyun Kim; Min Hye Yang; Jeong-Doo Heo; Sang Hyun Sung; Eun Ju Jeong

doi:10.20307/nps.2016.22.1.53

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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

Original Article

Natural Product Sciences 2016;22(1):53-59.

Published online: 17 December 2016

DOI: https://doi.org/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

Na-Hyun Kim¹, Min Hye Yang², Jeong-Doo Heo¹, Sang Hyun Sung³, Eun Ju Jeong^4,^∗

¹Gyeongnam Department of Environment & Toxicology, Korea Institute of Toxicology, 17 Jegok-gil, Munsan-eup, Gyeongnam 660-844, Republic of Korea

²College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea

³College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea

⁴ Department of Agronomy & Medicinal Plant Resources, College of Life Sciences and Natural Resources, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea

^∗Author for correspondence Eun Ju Jeong, Department of Agronomy & Medicinal Plant Resources, College of Life Sciences and Natural Resources, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea Tel: +82-55-751-3224; E-mail: ejjeong@gntech.ac.kr

Received 10 August 2015 Revised 16 September 2015 Accepted 16 September 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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Keywords: Euonymus alatus, Neolignan, Anti-inflammation, RAW264.7

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Fig. 1.

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

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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.

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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.

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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.

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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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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 (N^G-Nitro-L-arginine, a well known inhibitor of NOS) and L-NMMA (N^G-methyl-L-arginine acetate, an inhibitor of iNOS-induced nitric oxide production) were used as positive controls.

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