Rapid Identification and Systematic Mechanism of Flavonoids from Potentilla freyniana Bornm. Based on UHPLC-Q-Exactive Orbitrap Mass Spectrometry and Network Pharmacology

Potentilla freyniana Bornm. (P. freyniana), belonging to the family Rosaceae, has been used as a folk medicine in China. However, as we know, the constituents and the systematic elucidation of the mechanism were not fully investigated. Therefore, it is necessary to develop a rapid method using LC-MS and network pharmacology for the detection and identification of constituents and the systematic mechanism of P. freyniana. Firstly, the flavonoids were detected and identified based on ultra-high-performance liquid chromatography coupled with Quadrupole-Exactive Focus Orbitrap MS (UHPLC-Q-Exactive Orbitrap MS). After that, the potential targets of those constituents were obtained by database mining. Then, the core targets were predicted by protein-protein interaction network and network analysis. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out via DAVID. This finding revealed that P. freyniana possessed 43 flavonoids (40 of them were first reported) with 23 core target genes, which are associated with PI3K-Akt, MAPK, TNF signaling pathway, and pathway in cancer. This study demonstrated the multicompound, multitarget, and multimechanism of P. freyniana, which are very beneficial to develop the further study and utilization of this plant including the material basis and quality control research.


Introduction
Potentilla freyniana Bornm. (P. freyniana), a genus Potentilla of the family Rosaceae, named Difengzi, is a perennial plant with branched and tufted roots widely distributed and cultivated allaround of China, especially in Hunan, Hubei, Jiangxi. eir roots have been used as a folk medicine in clearing away heat and toxic materials for treating canker, bone tuberculosis, external bleeding [1][2][3]. Previous investigations on P. freyniana showed the presence of different compounds including terpenes and flavonoids [4][5][6][7] and possessed a variety of activities such as anti-inflammatory, analgesic effects [8,9]. However, as we know, the constituents and systematic pharmacological mechanism were not fully investigated. For instance, 14 compounds, including eriodictyol, phlorizin, were separated from the roots of P. freyniana [7]. erefore, it is worthwhile to establish a highly sensitive method for characterizing their chemical constituents and elucidating systematic pharmacological mechanism of P. freyniana. e complexity of chemicals in Traditional Chinese Medicine (TCM) including P. freyniana has presented a significant challenge in the rapid identification and characterization of components. During the past decades, HPLC-MS, as a new technique has been used to profile and identify the chemical in TCM due to its validity, sensitivity, and specialness [10,11]. Especially, UHPLC-HRMS such as UHPLC-Q-Exactive Orbitrap MS, UHPLC-Q-TOF MS, and UHPLC-LTQ-Orbitrap-MS, affording a higher and faster separation and higher resolution of mass, was a much more powerful equipment in the identification of TCM compared to traditional HPLC-MS [12][13][14].
Network pharmacology is an impressive methodology for investigating the systematic pharmacological mechanism through the constructing and analyzing biological networks such as protein-protein interaction, chemical-target-pathway network, which could provide direction for the further discovery of new drug without enormous time, money, and effort [15][16][17]. erefore, this current study was designed to develop a fast and effective method for the chemical characterization and systematic pharmacological mechanism of P. freyniana using UHPLC-Q-Exactive Orbitrap MS and network pharmacology. P. freyniana possessed 43 flavonoids (40 of them was first reported) with 23 core target genes, which are associated with PI3K-Akt, MAPK, TNF signaling pathway, and pathway in cancer. is study demonstrated the multicompound, multitarget, and multimechanism of P. freyniana, which are very beneficial for the further study and utilization of this plant including the material basis and quality control research.
Acetonitrile and methanol of chromatography grade were provided by MERCK (Darmstadt, Germany); e ultrapure water was produced by a milli-Q water purification system (Millipore, Milford, MA, United States); formic acid of LC-MS grade and all other reagents of analytical grade were purchased from Aladdin Industrial Corporation.
P. freyniana was collected from Tong-Dao country of Huaihua (109.86 longitude, 26.03 latitude), Hunan province and were identified by Professor Wei Cai (Hunan Provincial Key Laboratory of Dong Medicine, Hunan University of Medicine). e voucher specimen was deposited at School of Pharmaceutical Sciences, Hunan University of Medicine.
All LC-MS n analyses were performed on the Q-Exactive Focus Orbitrap MS connected to the UHPLC system via a heated electrospray ionization source ( ermo Electron, Bremen, Germany). e optimized tune operating parameters in negative ion mode were listed as follows: sheath gas and auxiliary gas flow rate of 30 and 10 arbitrary, respectively; the capillary and auxiliary gas heater temperatures of 320°C and 350°C, respectively; spray voltage of 3.0 kV; RF lens of 50; High-resolution MS analysis was performed at full scan MS 1 with the mass range of m/z 100-1000 at a resolution of 35000 and targeted MS 2 at a resolution of 17500 triggered by parallel reaction monitoring mode; nitrogen was set as sheath, auxiliary, and collision gas; the isolation widow was 2 amu, and the normalized collision energy (NCE) was 30%.

Data Processing and Analysis.
All high-resolution MS data were acquired and processed using the Xcalibur version (2.0 software, ermo Fisher Scientific, San Jose, CA, USA). e compounds were detected by the Compound Discover version 3 using the metabolism workflow templates by the expected compounds predicted method [18]. e detailed parameters of the workflow template were set as follows: the minimum peak intensity was set as 10000; the maximum element counts were C30 H60 O20; the mass tolerance of MS and MS 2 was within 5 and 10 ppm, respectively; baicalein and phloretin were set as the carbon skeleton; reduction, oxidation was set as Phase I transformation; glucoside conjugation, glucuronide conjugation, pentoside conjugation, methylation was set as Phase II transformation.

Target Identification of Flavonoids.
TCMSP database is a free and online database for potential target identification of small molecules, especially TCM. e target genes were converted to the official gene symbol by STRING (https:// string-db.org) or Uniport (https://www.uniprot.org).

Protein-Protein Interaction
Network. STRING was a free tool, which can construct the PPI network by uploading the potential targets. e species was set as "Homo sapiens" with a confidence score >0.4. e network analysis was performed at Cytoscape to obtain the core targets.

2.7.
GeneMANIA Analysis. GeneMANIA (http:// genemania.org) is an online free and friendly tool for investigating gene function and gene interaction. e species was set as "Homo sapiens."

GO and Pathway Analysis.
e GO and KEGG pathway analysis was performed on the DAVID (https://david. ncifcrf.gov, Version 6.8).
e specific species in the list and background was set as "Homo sapiens." e entire compounds, targets, and pathway network were visualized by Cytoscape.

Analytical Strategy.
In order to identify flavonoids fully, an analytical strategy based on UHPLC Q-Exactive Focus Orbitrap MS was established in this study. First, the sample was prepared and injected into the UHPLC Q-Exactive Focus Orbitrap MS to gain the full scan high-resolution MS data.
en, those data were processed using Compound Discover software with metabolism workflow to predict and detect the molecule of flavonoids.
ird, the MS 2 of the predicted molecule were acquired using UHPLC Q-Exactive Focus Orbitrap MS by parallel reaction monitoring mode. Finally, the compounds were identified based on the full scan MS, MS 2 data, retention time, and bibliography.

Identification of Flavonoids.
e total content of flavonoids was measured by NaNO 2 -Al(NO 3 ) 3 -NaOH spectrophotometric colorimetry [19]. e calibration curve obtained by the rutin standard of absorbance concentrations(mg/mL) using five dilutions was y � 7.15x − 0.001, with the corresponding determination coefficient at 0.9999. Finally, the content of flavonoids is 32.17 ± 0.26%. A total of 43 constituents were unanimously and tentatively characterized based on UHPLC Q-Exactive Focus Orbitrap MS combined with the expected compounds predicted method. 40 excluded eriodictyol, phloretin, and hyperoside were reported from P. freyniana for the first time. e detailed information of those compounds is listed in Table 1. e high-resolution extracted ion chromatography is shown in Figure 1.

GeneMANIA
Analysis. Among the 23 key target genes and their interacting genes, it was found that 42.75 % had coexpression characteristics, 41.10 % displayed physical interactions characteristic. Other characteristics, including pathway, genetic interactions, colocalization, and shared protein domains, are displayed in Figure 2. 3.6. GO and Pathway Analysis. In order to further study the 23 core target genes, GO and KEGG pathway analysis were performed by DAVID. GO term enrichment analysis results were divided into the biological process (BP, 23/23), cell compound (CC, 23/23), and molecular function (MF, 23/ 23). A total of 158 BP, 16 CC, and 33 MF has a p-value less International Journal of Analytical Chemistry 5 than 0.05 (Table S2). In GO term enrichment analysis, the BP might be related to positive regulation of transcription from RNA polymerase II promoter (10/23), response to drug (9/23), negative regulation of apoptotic process ( Figure 3. In addition, 83 KEGG pathways (Table S3)

Network Analysis.
Based on the target and KEGG pathway analysis, the entire compounds, targets, and pathway network were established by Cytoscape. e network with 122 nodes and 595 edges is shown in Figure 5. e red diamond, green ellipse, and blue triangle represent compounds, genes, and pathways, respectively.

Conclusion
In the present investigation, this finding revealed that P. freyniana possessed 43 flavonoids (40 of them was first reported) with 23 core target genes, which were associated with PI3K-Akt, MAPK, TNF signaling pathway, and pathway in cancer. is study demonstrated the multicompound, multitarget, and multimechanism of P. freyniana, which are very beneficial for the further study and utilization of this plant including the material basis and quality control research.

Data Availability
e data used to support the finding of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.  Figure 5: Chemical-target genes-pathway network: red represents chemical; blue represents target genes; green represents pathway. 8 International Journal of Analytical Chemistry