Rapid screening of neuraminidase inhibitors with the benzoic acid skeleton from Paeonia suffruticosa Andrews by solid-phase extraction with an enzyme activity switch combined with mass spectrometry analysis
Introduction
Finding active ingredients from natural herbs is a very important way of drug development [1,2]. The bioactivity and draggability of compounds are closely related to the skeleton structures [3,4]. The discovery of active compounds with specific skeletons can reduce the blindness of lead compound discovery and improve the efficiency of drug development. The traditional method for the finding of specific skeleton compounds with bioactivities was isolation followed by biological assays. This strategy has unique advantages in the identification of bioactive compounds with certain skeletons and unreported skeletons [2,[4], [5], [6]]. However, it contains many steps, including sample extraction, isolation, purification, structure identification, and biological assays. It was not suitable for rapid screening. A simpler and faster strategy is urgently needed in the screening of bioactive compounds with certain skeletons. As a common sample processing technique in the laboratory, solid-phase extraction (SPE) is often used in the accumulation of natural products [7], [8], [9], [10], because of its simple and rapid operations. Considering this, it is attractive to apply the SPE in the screening of bioactive compounds with specific skeletons from natural resources.
As an illustrative example, neuraminidase (NA) and the root cortex of Paeonia suffruticosa Andrews (CPSA) were used as the biotarget and medicinal herb to screen the NA inhibitors with the specific skeleton. NA is a tetramer glycoprotein on the envelope of the influenza virus and plays an important role in the transmission of the virus [11], [12], [13]. One of the research hotspots of NA inhibitors is benzoic acid-derived compounds because of their simple synthesis process, simple stereochemical structure, and high bioavailability [14], [15], [16], [17]. Benzoic acid-derived inhibitors (BAIs) of NA are mainly chemo-synthesized molecules based on rational drug design, and limited numbers have been discovered from natural herbs. CPSA is a well-known medicinal herb for its effects of activating blood circulation, dissipating blood stasis, curing carbuncle and sore poison [18,19].
In this study, the SPE with an EA switch combined with mass spectrometry analysis was proposed (Fig. 1). The enzyme was incubated with the crude extract solution forming enzyme-inhibitor complexes at first. The SPE was used to separate the sample solution based on polarities. The EA switch was designed to detect and collect the fractions containing enzyme-inhibitor complexes and could exclude most of the non-target compounds. And the mass spectrometry analysis was used to pick out the specific skeleton inhibitors from the collected fractions.
As a proof-of-concept, the BAIs of NA were obtained from CPSA in three steps. Firstly, NA and CPSA crude extracts were incubated to get the sample solution containing two types of enzyme-inhibitor complexes (NA-BAIs and NA-Is), NA, and small molecules with different polarities (LP1, LP2, SP). The enzyme-inhibitor complexes and enzymes are with large polarity and could dissolve in the mobile phase. Next, the sample was separated, detected, and collected by the SPE with an EA switch. Molecules with large polarity (NA-BAIs, NA-Is, NA, LP1) were eluted first, and they were detected and collected simultaneously by the EA switch. When the detected values reduced significantly, the six-way valve of the EA switch was triggered to end the collection process. The medium and small polarity compounds (LP2, SP) flowed into the waste bottle. The collected eluents were treated for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Finally, the fragment pathways of benzoic acid-derived standard compounds were used as the guidance for the pinpointing of BAIs from the collected eluents. Two natural benzoic acid derived NA inhibitors were successfully identified. In this study, the complicated isolation and purification were avoided, and the separation, detection, and collection were carried out on one instrument system. In addition to the traditional use of SPE, we first applied the SPE with an EA switch combined with mass spectrometry analysis in finding the bioactive compounds with specific skeletons.
Section snippets
Reagents and materials
The dried root cortex of Paeonia suffruticosa Andrews was purchased from De Ren Tang drugstore (Jinan, Shandong province, China). Voucher specimens (No. CPSA202103) were deposited at the College of Medicine, Shandong University of Traditional Chinese Medicine.
NA from C. perfringens and standard compounds (paeoniflorin, oxypaeoniflorin, gallic acid, vanillic acid, benzoic acid, p-hydroxybenzoic acid, methyl vanillic acid) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai,
The design and trigger of the SPE with an EA switch
An EA switch was designed to control the collection process of the sample. The switch was simply assembled by three parts, 96-well plates, a fluorescence detector, and a six-way valve (the dashed part in Fig. 2). To find the trigger signal of the switch, the NA solution was used as the sample and was loaded into the SPE cartridge. The MES buffer containing 2% methanol was used as the mobile phase. The eluents were collected every 15 seconds, and the optical density (OD) values of the eluents
Conclusions
Overall, SPE with an EA switch combined with mass spectrometry analysis as a novel strategy was proposed for the precise screening of benzoic acid skeleton-based neuraminidase inhibitors from CPSA. The operations of separation, detection, and collection were carried out on one instrument system. Using this strategy, two benzoic acid derivatives, paeoniflorin, and oxypaeoniflorin were successfully discovered as a new class of natural NA inhibitors. Promisingly, this strategy could provide an
CRediT authorship contribution statement
Menghan Chen: Conceptualization, Data curation, Investigation, Formal analysis, Methodology, Writing – original draft. Liqing Wang: Investigation, Formal analysis. Shuyan Xing: Formal analysis, Investigation. Yong Yang: Supervision, Resources. Rong Rong: Supervision, Resources, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grant numbers 81873220, 81774167); and the Key R & D plan of Shandong Province (major science and technology innovation project) (grant number 2020CXGC010505).
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