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Identification of nuclear factor-κB inhibitors in the folk herb Rhizoma Menispermi via bioactivity-based ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis.

Sun D, Zhou M, Ying X, Cheng B, Han Y, Nie Y, Hou Y, Bai G - BMC Complement Altern Med (2014)

Bottom Line: Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which norsinoacutin, 6-O-methyl-laudanosoline-13-O-glucopyranoside laurifloline, dauricinoline and N-norsinoacutin-β-D-glucopyranoside were revealed as new NF-κB inhibitors.This method of UPLC-Q/TOF coupled with the luciferase reporter assay system was initially applied to the study of RM and was demonstrated to represent a simple, rapid and practical approach to screen for anti-inflammatory compounds.This study provided useful results for further investigation on the anti-inflammatory mechanism of RM.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China. houyy@nankai.edu.cn.

ABSTRACT

Background: Rhizoma Menispermi (RM) is the dried root of Menispermum dauricum DC, which is traditionally used to treat swelling and pain for sore throat, enteritis and rheumatic arthralgia in the clinic, but its bioactive compounds remain unclear.

Methods: In this study, RM extract was administered orally to ICR mice followed by challenging with an intratracheal Pseudomonas aeruginosa suspension. Then mortality, histological features of lung, and inflammatory cytokines were evaluated. RM treatment significantly ameliorated Pseudomonas aeruginosa-induced acute lung inflammation and reduced levels of inflammatory mediators. To screen for potential anti-inflammatory constituents of the RM extract, a simple and rapid method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) coupled with a luciferase reporter assay system to detect nuclear factor-κB (NF-κB) activity was established.

Results: Using this system, seven potential NF-κB inhibitors were detected, including sinomenine, norsinoacutin, N-norsinoacutin-β-D-glucopyranoside, 6-O-methyl-laudanosoline-13-O-glucopyranoside, magnoflorine, laurifloline and dauricinoline. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which norsinoacutin, 6-O-methyl-laudanosoline-13-O-glucopyranoside laurifloline, dauricinoline and N-norsinoacutin-β-D-glucopyranoside were revealed as new NF-κB inhibitors.

Conclusion: This method of UPLC-Q/TOF coupled with the luciferase reporter assay system was initially applied to the study of RM and was demonstrated to represent a simple, rapid and practical approach to screen for anti-inflammatory compounds. This study provided useful results for further investigation on the anti-inflammatory mechanism of RM.

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Related in: MedlinePlus

Confirmation of the effects by potential NF-kB inhibitors.A: Effects of the potential NF-kB inhibitors on the NF-kB expression. B and C: IL-6 and IL-8 expression in TNF-α induced BEAS-2B cells, respectively. Each bar represents the mean + SEM; n = 4 for each group. (1: N-norsinoacutin-β-Dglucopyranoside; 2: 6-O-methyl-laudanosoline-13-O-glucopyranoside; 3: sinomenine; 4: norsinoacutin; 5: magnoflorine; 6: laurifloline; 7: dauricinoline). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the model group. ##p < 0.01 compared to the control group.
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Fig5: Confirmation of the effects by potential NF-kB inhibitors.A: Effects of the potential NF-kB inhibitors on the NF-kB expression. B and C: IL-6 and IL-8 expression in TNF-α induced BEAS-2B cells, respectively. Each bar represents the mean + SEM; n = 4 for each group. (1: N-norsinoacutin-β-Dglucopyranoside; 2: 6-O-methyl-laudanosoline-13-O-glucopyranoside; 3: sinomenine; 4: norsinoacutin; 5: magnoflorine; 6: laurifloline; 7: dauricinoline). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the model group. ##p < 0.01 compared to the control group.

Mentions: The activities of the bioactive compounds (100 μmol/L) in RM were evaluated using the dual-luciferase reporter assay system. As shown in Figure 5A, Dex (10 μmol/L) and the seven potential NF-κB inhibitors (ingredients of RM) displayed significant NF-κB inhibitory effects (p < 0.05) compared to the model group. Especially, sinomenine, magnoflorine and laurifloline (p < 0.001) displayed highly significant inhibition. Because NF-κB transcription factors synergistically activate transcription of the inflammatory cytokines IL-6 and IL-8, the anti-inflammatory effect of these seven ingredients on IL-6 and IL-8 were further evaluated [32]. As shown in Figure 5B, IL-6 expression was reduced by six of the compounds (p < 0.05), highly significantly by laurifloline and sinomenine (p < 0.001); however, norsinoacutin displayed no significant IL-6 inhibition based on our assay. For IL-8, as shown in Figure 5C, sinomenine, norsinoacutin, magnoflorine and laurifloline demonstrated an inhibitory effect (p < 0.05), but the other three compounds did not display an inhibitory effect. Dex (10 μmol/L), the positive control, significantly inhibited both IL-6 (p < 0.001) and IL-8 expression (p < 0.05). The results further confirmed the anti-inflammatory effects of the seven compounds as NF-κB inhibitors in RM.Figure 5


Identification of nuclear factor-κB inhibitors in the folk herb Rhizoma Menispermi via bioactivity-based ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis.

Sun D, Zhou M, Ying X, Cheng B, Han Y, Nie Y, Hou Y, Bai G - BMC Complement Altern Med (2014)

Confirmation of the effects by potential NF-kB inhibitors.A: Effects of the potential NF-kB inhibitors on the NF-kB expression. B and C: IL-6 and IL-8 expression in TNF-α induced BEAS-2B cells, respectively. Each bar represents the mean + SEM; n = 4 for each group. (1: N-norsinoacutin-β-Dglucopyranoside; 2: 6-O-methyl-laudanosoline-13-O-glucopyranoside; 3: sinomenine; 4: norsinoacutin; 5: magnoflorine; 6: laurifloline; 7: dauricinoline). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the model group. ##p < 0.01 compared to the control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4195958&req=5

Fig5: Confirmation of the effects by potential NF-kB inhibitors.A: Effects of the potential NF-kB inhibitors on the NF-kB expression. B and C: IL-6 and IL-8 expression in TNF-α induced BEAS-2B cells, respectively. Each bar represents the mean + SEM; n = 4 for each group. (1: N-norsinoacutin-β-Dglucopyranoside; 2: 6-O-methyl-laudanosoline-13-O-glucopyranoside; 3: sinomenine; 4: norsinoacutin; 5: magnoflorine; 6: laurifloline; 7: dauricinoline). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the model group. ##p < 0.01 compared to the control group.
Mentions: The activities of the bioactive compounds (100 μmol/L) in RM were evaluated using the dual-luciferase reporter assay system. As shown in Figure 5A, Dex (10 μmol/L) and the seven potential NF-κB inhibitors (ingredients of RM) displayed significant NF-κB inhibitory effects (p < 0.05) compared to the model group. Especially, sinomenine, magnoflorine and laurifloline (p < 0.001) displayed highly significant inhibition. Because NF-κB transcription factors synergistically activate transcription of the inflammatory cytokines IL-6 and IL-8, the anti-inflammatory effect of these seven ingredients on IL-6 and IL-8 were further evaluated [32]. As shown in Figure 5B, IL-6 expression was reduced by six of the compounds (p < 0.05), highly significantly by laurifloline and sinomenine (p < 0.001); however, norsinoacutin displayed no significant IL-6 inhibition based on our assay. For IL-8, as shown in Figure 5C, sinomenine, norsinoacutin, magnoflorine and laurifloline demonstrated an inhibitory effect (p < 0.05), but the other three compounds did not display an inhibitory effect. Dex (10 μmol/L), the positive control, significantly inhibited both IL-6 (p < 0.001) and IL-8 expression (p < 0.05). The results further confirmed the anti-inflammatory effects of the seven compounds as NF-κB inhibitors in RM.Figure 5

Bottom Line: Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which norsinoacutin, 6-O-methyl-laudanosoline-13-O-glucopyranoside laurifloline, dauricinoline and N-norsinoacutin-β-D-glucopyranoside were revealed as new NF-κB inhibitors.This method of UPLC-Q/TOF coupled with the luciferase reporter assay system was initially applied to the study of RM and was demonstrated to represent a simple, rapid and practical approach to screen for anti-inflammatory compounds.This study provided useful results for further investigation on the anti-inflammatory mechanism of RM.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China. houyy@nankai.edu.cn.

ABSTRACT

Background: Rhizoma Menispermi (RM) is the dried root of Menispermum dauricum DC, which is traditionally used to treat swelling and pain for sore throat, enteritis and rheumatic arthralgia in the clinic, but its bioactive compounds remain unclear.

Methods: In this study, RM extract was administered orally to ICR mice followed by challenging with an intratracheal Pseudomonas aeruginosa suspension. Then mortality, histological features of lung, and inflammatory cytokines were evaluated. RM treatment significantly ameliorated Pseudomonas aeruginosa-induced acute lung inflammation and reduced levels of inflammatory mediators. To screen for potential anti-inflammatory constituents of the RM extract, a simple and rapid method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) coupled with a luciferase reporter assay system to detect nuclear factor-κB (NF-κB) activity was established.

Results: Using this system, seven potential NF-κB inhibitors were detected, including sinomenine, norsinoacutin, N-norsinoacutin-β-D-glucopyranoside, 6-O-methyl-laudanosoline-13-O-glucopyranoside, magnoflorine, laurifloline and dauricinoline. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which norsinoacutin, 6-O-methyl-laudanosoline-13-O-glucopyranoside laurifloline, dauricinoline and N-norsinoacutin-β-D-glucopyranoside were revealed as new NF-κB inhibitors.

Conclusion: This method of UPLC-Q/TOF coupled with the luciferase reporter assay system was initially applied to the study of RM and was demonstrated to represent a simple, rapid and practical approach to screen for anti-inflammatory compounds. This study provided useful results for further investigation on the anti-inflammatory mechanism of RM.

Show MeSH
Related in: MedlinePlus