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. 2019 May 8;24(9):1789.
doi: 10.3390/molecules24091789.

Robustaflavone Isolated from Nandina domestica Using Bioactivity-Guided Fractionation Downregulates Inflammatory Mediators

Ara Jo  1 , Hyun Ji Yoo  2 , Mina Lee  3
Affiliations

Robustaflavone Isolated from Nandina domestica Using Bioactivity-Guided Fractionation Downregulates Inflammatory Mediators

Ara Jo et al. Molecules. .

Abstract

Nandina domestica (Berberidaceae) has been used in traditional medicine for the treatment of cough. This plant is distributed in Korea, Japan, China, and India This study aimed to investigate the anti-inflammatory phytochemicals obtained from the N. domestica fruits. We isolated a biflavonoid-type phytochemical, robustaflavone (R), from N. domestica fruits through bioactivity-guided fractionation based on its capacity to inhibit inflammation. The anti-inflammatory mechanism of R isolated from N. domestica has not yet been studied. In the present study, we evaluated the anti-inflammatory activities of R using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We have shown that R reduces the production of nitric oxide (NO), pro-inflammatory cytokine interleukin-1 beta (IL-1β), and IL-6. Western blot analysis showed that R suppresses the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and downregulates the expression of LPS-induced nuclear factor-kappa B (NF-κB) and the phosphorylation of extracellular-regulated kinases (pERK 1/2). Moreover, R inhibited IL-8 release in LPS-induced human colonic epithelial cells (HT-29). These results suggest that R could be a potential therapeutic candidate for inflammatory bowel disease (IBD).

Keywords: Nandina domestica; bioactivity-guided isolation; inflammatory bowel disease; inflammatory mediators; robustaflavone.

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Conflict of interest statement

The authors have no conflict of interest to disclose.

Figures

Figure 1
Figure 1
Effects of the total extract and extract fractions of Nandina domestica fruits on cell viability (A) and nitric oxide production (B). RAW 264.7 cells were cultured in the presence of the extracts for 1 h and stimulated with lipopolysaccharide (LPS) (1 µg/mL) for 16 h. Cell viability and NO production were detected using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and the Griess reagent, respectively. Nitrite concentrations from the non-treated and LPS-treated controls were 0.81 ± 0.1 μM and 17.2 ± 0.2 μM, respectively. Each determination was made in triplicate. The data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
Figure 2
Figure 2
Effects of ethyl acetate (EtOAc) subfractions (E1–E5) on cell viability (A) and NO production (B). RAW 264.7 cells were cultured in the presence of the N. domestica extract for 1 h and stimulated with LPS (1 µg/mL) for 16 h. Cell viabilities and NO production were detected using the MTT assay and the Griess reagent, respectively. Nitrite concentrations of non-treated and LPS-treated controls were 0.8 ± 0.04 μM and 17.5 ± 0.3 μM, respectively. Each experiment was performed in triplicate. The data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
Figure 3
Figure 3
Schematic representation of the isolation of robustaflavone (R) from N. domestica fruits using bioactivity-guided fractionation. Bioactivity-guided fractionation of N. domestica fruit was performed as shown in the schematic representation and resulted in the isolation and identification of R. Fractionation was guided by assessing the inhibitory effect of R on NO production at concentrations of 100 μg/mL without any cytotoxicity. At each level of fractionation, all the fractions generated were tested simultaneously and were compared to the crude extract.
Figure 4
Figure 4
Effects of R on cell viability (A) and NO production (B). RAW 264.7 cells were treated with R for 1 h and stimulated with LPS (1 µg/mL) for 16 h. Cell viability and NO production were detected using the MTT assay and Griess reagent, respectively. Nitrite concentrations of non-treated and LPS-treated controls were 0.8 ± 0.1 μM and 17.3 ± 0.2 μM, respectively. Each experiment was performed in triplicate. The data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
Figure 5
Figure 5
Effects of R on iNOS and cyclooxygenase COX-2 expression. RAW 264.7 cells were incubated in the presence of R for 1 h and then stimulated with LPS (1 µg/mL) for 16 h. The expression of iNOS (A), COX-2 (B), and β-actin in the LPS-induced cells was determined by Western blot analysis. The relative density was calculated as the ratio of the level of each protein expressed to the level of β-actin. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
Figure 6
Figure 6
Effect of R on the expression of proinflammatory cytokines, IL-1β and IL-6. RAW 264.7 cells were pretreated with R for 1 h, then incubated with LPS (1 µg/mL) for 16 h. The levels of IL-1β (A) and IL-6 (B) in culture media were measured by ELISA. The data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
Figure 7
Figure 7
Effect of R on NF-κB activation (A) and pERK1/2 expression (B). RAW 264.7 cells were cultured in the presence of R for 16 h and stimulated with LPS (1 µg/mL) for 1 h under serum-free conditions. The expression of NF-κB, p65, and pERK1/2 was detected by Western blot analysis. The results presented are representative of three independent experiments. The relative density was calculated as the ratio of the expression level of each protein with that of β-actin. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-treated group.
Figure 8
Figure 8
Effect of R on IL-8 expression. HT-29 colon epithelial cells were pretreated with R for 2 h and stimulated with LPS (100 ng/mL) for 12 h. The levels of IL-8 in the culture media were measured by ELISA. The data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, vs. LPS-treated group.
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References

    1. Ahmed A.U. An overview of inflammation: Mechanism and consequences. Front. Biol. 2011;4:274–281. doi: 10.1007/s11515-011-1123-9. - DOI
    1. Spranger J., Kroke A., Mohlig M., Hoffmann K., Bergmann M.M., Ristow M., Boeing H., Pfeiffer A.F.H. Inflammatory Cytokines and the Risk to Develop Type 2 Diabetes. J. Diabetes. 2003;52:814–817. doi: 10.2337/diabetes.52.3.812. - DOI - PubMed
    1. Park J.Y., Chung T.W., Jeong Y.J., Kwak C.H., Ha S.H., Kwon K.M., Abekura F., Cho S.H., Lee Y.C., Ha K.T., et al. Ascofuranone inhibits lipopolysaccharide–induced inflammatory response via NF-kappaB and AP-1, p-ERK, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages. PLoS ONE. 2017;10:e0171322. doi: 10.1371/journal.pone.0171322. - DOI - PMC - PubMed
    1. Mitchell J.A., Larkin S., Williams T.J. Cyclooxygenase-2: Regulation and relevance in inflammation. Biochem. Pharmacol. 1995;50:1535–1542. doi: 10.1016/0006-2952(95)00212-X. - DOI - PubMed
    1. Seok S.J., Jin U.H., Kim K.W., Son J.K., Lee S.H., Son K.H., Chang H.W., Lee Y.C., Kim C.H. Triterpenoid saponin, oleanolic acid 3-O-β-d-glucopyranosyl(1 → 3)-α-l-rhamnopyranosyl(1 → 2)-α-l-arabinopyranoside (OA) from Aralia elata inhibits LPS-induced nitric oxide production by down-regulated NF-κB in raw 264.7 cells. Arch. Biochem. Biophys. 2007;467:227–233. doi: 10.1016/j.abb.2007.08.025. - DOI - PubMed

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