Data_Sheet_1_Combining UPLC/Q-TOF-MS/MS With Biological Evaluation for NF-κB Inhibitors in Uyghur Medicine Althaea rosea Flowers.docx

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.</p

Preparation of Functionalized Alkynyl Magnetic Microspheres for the Selective Enrichment of Cell Glycoproteins Based on Click Chemistry

Functionalized alkynyl polyvinyl alcohol magnetic microspheres (PVA MMs) were developed for the specific enrichment of sialic acid-rich glycoproteins by click chemistry. The capture capability for proteins was evaluated through a novel dual-labeled bovine serum albumin (BSA) that utilizes fluorescence resonance energy transfer (FRET). The PVA MM parameters, including the size and coverage of functionalized groups, were optimized by response surface methodology. The optimal parameters obtained were 1.25–6.31 μm in size and 48.53–73.05% in coverage. Then, the optimal PVA MMs were synthesized, and the morphology and surface chemical properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). To capture glycoproteins from the cell surface, a bioorthogonal chemical method was applied to metabolically label them with an azide group. The functionalized alkynyl PVA MMs showed a high specificity and strong binding capability for glycoproteins through a [3 + 2] cycloaddition reaction. The results indicated that the functionalized alkynyl PVA MMs could be applied to the enrichment of cell glycoproteins, and the merits of the MMs suggested an attractive and potential way to facilitate glycoprotein research

Confirmation of the bioactive compounds from the <i>LP</i> in TNF-α-induced BEAS-2B cells.

<p>(<b>A</b>) Effects of the potential NF-κB inhibitors by the dual-luciferase reporter assay system, effects of the representatives of four NF-κB inhibitor types on (<b>B</b>) IL-6 and (<b>C</b>) IL-8 expression. Each bar represents the mean ± SEM. <i>n</i> = 5, **<i>p</i> < 0.05 <i>vs</i> the group that was treated with TNF-α in the absence of extracts and drugs, and ^##<i>p</i> < 0.05 <i>vs</i> the Control group. One (*) or two (**) asterisks indicate <i>p</i> < 0.05 or <i>p</i> < 0.01, respectively.</p

Effects of different organs of <i>LN</i> on NF-κB inhibition (A) and the heat map (B).

<p><b>A</b>: The levels of NF-κB in TNF-α stimulated HEK 293 cells. Values are presented as the mean ± SEM, <i>n</i> = 5 for each group. **<i>p</i> <0.05 <i>vs</i> the group that was treated with TNF-α in the absence of extracts and drugs, ^##<i>p</i> < 0.05 <i>vs</i> the Control group; <b>B</b>: The relative content of each marker ingredient in different organs. Zero content was expressed as red and largest content (100%) as green. </p

Results of PCA and the BPI chromatograms of different organs of <i>LN</i>.

<p>BPI chromatograms (<b>A</b>), score plots (<b>B</b>) and Loading plots (<b>C</b>) in the positive and negative ESI mode are shown. The peak numbers, significance values and relative content (MAX.) are consistent with those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081971#pone-0081971-t001" target="_blank">Table 1(A and B)</a>. </p

Chemical structures of the bioactive compounds in the <i>LP.</i>

<p>Chemical structures of the bioactive compounds in the <i>LP.</i></p

The organ-active-ingredients application for <i>LN</i> organs.

<p>The organ-active-ingredients application for <i>LN</i> organs.</p

UPLC-Q-TOF/MS and bioactivity analyses of <i>LP</i>.

<p>(<b>A</b>) UPLC-UV chromatograms of LP TIC data in (<b>B</b>) the positive and (<b>C</b>) negative ESI modes. Bioactivity chromatograms were obtained via the dual-luciferase reporter assay system for (D) NF-κB inhibition. NF-κB inhibition values are presented as the mean ± SEM, <i>n</i> = 5 for each group. The peak numbers are consistent with those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081971#pone-0081971-t001" target="_blank">Table 1(A and B)</a>. </p