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. 2019 Jan 9:9:1975.
doi: 10.3389/fpls.2018.01975. eCollection 2018.

Combining UPLC/Q-TOF-MS/MS With Biological Evaluation for NF-κB Inhibitors in Uyghur Medicine Althaea rosea Flowers

Fang Ma  1 , Qingxin Cui  1 , Gang Bai  1
Affiliations

Combining UPLC/Q-TOF-MS/MS With Biological Evaluation for NF-κB Inhibitors in Uyghur Medicine Althaea rosea Flowers

Fang Ma et al. Front Plant Sci. .

Abstract

The Althaea rosea (Linn.) flower is a common plant that is often used to control inflammation in Uyghur ethnic medicine. However, its active ingredients remain uncertain and difficult to identify, severely limiting its use as a valuable crop. This paper aims to establish a rapid assay strategy for the integration of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS) and a biologically active (NF-κB inhibitor) luciferase reporter detection system to explore various anti-inflammatory compounds of A. rosea (Linn.) flowers. Potential anti-inflammatory components were screened using the NF-κB activity assay system and simultaneously identified based on mass spectrometry data. Four structural types of NF-κB inhibitors (phenolic acid, hydroxycinnamic acid, flavonoid, and dihydroflavone) were identified. Further cytokine assays confirmed their potential anti-inflammatory effects as NF-κB inhibitors. Compared with traditional chromatographic separation, integrated UPLC/Q-TOF-MS/MS identification compounds, and biological activity verification are more convenient and more reliable. This strategy clearly demonstrates that fingerprinting based on MS data not only can identify unknown components but also is a powerful and useful tool for screening trace active ingredients directly from complex matrices. A. rosea (Linn.) exhibits great health and pharmaceutical value and may contribute to the development of new anti-inflammatory drugs.

Keywords: Althaea rosea (Linn.) flowers; NF-κB inhibitors; UPLC/Q-TOF-MS/MS; anti-inflammatory compounds; constituent identification.

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Figures

FIGURE 1
FIGURE 1
UPLC/Q-TOF-MS/MS and bioactivity analysis of the Althaea rosea flowers. (A) Total Ion Chromatography (TIC) chromatograms in positive ESI mode. (B) TIC chromatograms in negative ESI mode. (C) Bioactivity chromatograms obtained via the luciferase reporter assay system for NF-κB inhibition. The peak numbers are consistent with those reported in Table 1.
FIGURE 2
FIGURE 2
Chemical structures of the bioactive compounds in Althaea rosea flowers.
FIGURE 3
FIGURE 3
Confirmation of NF-κB inhibitors from Althaea rosea flowers by the luciferase reporter assay system. Each bar represents the mean ± SEM, n = 5 per group.
FIGURE 4
FIGURE 4
Determination of IC50 of representative compounds. (A) The inhibition curves of representative compounds. (B) The IC50 values of representative compounds.
FIGURE 5
FIGURE 5
Effects of representative compounds of NF-κB inhibitor on IL-6 and IL-8 expression in TNF-α-induced HEK 293 T. Error bars indicate SEM, n = 4. ∗∗p < 0.01 vs. model group; ∗∗∗p < 0.001 vs. model group; ###p < 0.001 vs. control group.
See this image and copyright information in PMC

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