Integrated UPLC-Q-TOF-MS/MS and Network Pharmacology Approach to Investigating the Metabolic Profile of Marein of Coreopsis tinctoria Nutt.

Marein is the main active compound of Coreopsis tinctoria Nutt., and its main activities include antioxidant, hypoglycemic, and hypotensive. After oral administration of marein, the blood concentration of marein is low. The metabolites of marein have not been reported systematically. In this study, a rapid and systematic method based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was established to detect metabolites of marein in vivo (plasma and urine) after oral administration and injection. Sixty-one metabolites were identified. The metabolites are formed through a wide range of metabolic reactions, including hydroxylation, glucuronidation, methylation, hydrolysis, and desorption of hydrogen. The liver microsome incubation was further used to investigate the metabolic rate of marein. Network pharmacology was applied to study the targets and pathways of marein and its metabolites. Marein and its metabolites act on the same targets to enhance the therapeutic effect. This research illuminates the metabolites and metabolic reaction of marein and establishes a basis for the development and rational utilization of C. tinctoria. Meanwhile, the analysis of prototype and metabolites together by network pharmacology techniques could provide a methodology for the study of component activity.

Marein, the glucoside of chalcone, is the main active component of flavonoids in C. tinctoria [16][17][18]. It has effects on lowering blood sugar, regulating blood lipids, and anti-hypertension [3]. Experiments in vitro have proved that marein can resist oxidation [19,20], reduce triacylglycerol content, and have a certain protective effect on pancreatic islet cells MIN6 [11]. Marein also improves glucose metabolism disorder induced by high glucose in HepG2 cells, which could significantly prevent insulin resistance induced by high glucose [21,22]. And marein can improve the abnormal glucose and lipid metabolism of hypertrophic H9c2 cells by reducing the expression of HIF-1α [23]. It also prevents diabetic nephropathy by inhibiting the expression of SGLT2 in the kidney [24].
However, after healthy volunteers drank a tea of C. tinctoria, no marein is detected in their blood [21]. In the preliminary experiments of our team, after intragastric administration in rats, no marein was found in plasma. e low blood concentration and poor bioavailability of marein make it difficult to explain the remarkable curative effect. erefore, a study of the metabolic processes and pharmacological action of metabolites of marein are needed.
After oral administration, natural products take effects in vivo based on the combination of prototype and metabolites. Network pharmacology, as a powerful tool for analysis of multicomponent effects [25,26], reveals the complex biological network relationships among compounds, genes, and targets by using network visualization techniques to analyze the behavior of compounds acting on different targets, cells, and organs at the molecular and gene levels. Using the network pharmacology method, the "compound-targetpathway" network can be constructed according to the structure and efficacy of compounds, which can effectively predict the activities and mechanism of components.
In this article, a specific and sensitive ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method was established for rapid screening and systematic identification of metabolites of marein. e oral and injected metabolites of marine were identified in biological samples including plasma and urine. en, an in vitro incubation system of rat liver microsomes was established. Network pharmacology was used to study the component-target-pathway network of marein and its metabolites. e research establishes a scientific basis for the development and rational use of C. tinctoria.

Animal Handling and Specimen Collection.
Forty-eight healthy male Sprague-Dawley rats, SPF grade, weighing 250 ± 10 g, were purchased from Huafukang Laboratory Animal Technology Co. Ltd. (Beijing, China). Before use, all animals were reared for 5 days at a constant temperature of 25°C, a humidity of 40-60%, and a 12-hour light-dark cycle. All experimental protocols strictly followed the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978), the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China (2006), and the Animal Use and Care Committee of Tianjin University of Traditional Chinese Medicine (No. IRMDWLL-201903).

Oral Administration.
After adaptive feeding for 1 week, 24 SD rats were randomly divided into 4 groups: blank blood sample group, blank urine group, marein blood sample group, and marein urine group. e experimental animals were fasted 12 hours before the administration and free drinking water. Marein was dissolved with an appropriate amount of CMC-Na solution and was administered to SD rats by gavage at a dose of 200 mg/kg. e blank blood sample group was given an equal volume of CMC-Na solution.

Administration by Injection.
After adaptive feeding of 24 SD rats for 1 week, they were randomly divided into 4 groups, namely: blank blood sample group, blank urine group, marein blood sample group, and marein urine group. e experimental animals were fasted 12 hours before the administration and free drinking water. Marein was dissolved in appropriate amounts of saline and administered to SD rats via tail vein injection at a dose of 10 mg/kg. e blank group was injected with an equal volume of saline through the tail vein.

Plasma Sample
Collection. Blood samples were collected from the fundus venous plexus of rats at 0, 0.08, 0.25, 0.5, and 1 h before and after gavage and injection. e blood samples were centrifuged (9,900 g, 10 min) to obtain plasma samples. All plasma samples were stored at − 80°C until they were processed and analyzed.

Urine Samples Collection.
Urine samples were collected for 24 hours by gavage and injection in a metabolic cage. All urine samples were stored at − 80°C until they were processed and analyzed.

Sample Preparation.
Take 100 μL of plasma from each group at the same time point and combine, add 900 μL of acetonitrile, vortex for 3 min, and centrifuge (4°C, 19,500 g) for 10 min. Take the supernatant, blow-dry, and concentrate and then add 900 μL of acetonitrile. Vortex for 3 minutes and then centrifuge (4°C, 19,500 g) for 10 minutes. Take the supernatant, dry it with nitrogen, and concentrate. Add 100 μL of 50% methanol to reconstitute the residue (methanol:water � 1:1), vortex, and centrifuge (4°C, 19,500 g) for 10 minutes. e supernatant was taken and analyzed by UPLC-Q-TOF-MS/ MS. e blank plasma sample was processed in the same way.
Take 400 μL of urine, add 800 μL of methanol, vortex for 3 minutes, and centrifuge (4°C, 19,500 g) for 10 minutes. Take the supernatant, blow-dry, and concentrate and then add 800 μL of methanol to reconstitute, vortex for 3 minutes, and centrifuge (4°C, 19,500 g) for 10 minutes. In parallel, the supernatants were combined, dried, concentrated with nitrogen, and finally reconstituted with 100 μL 50% methanol (methanol:water � 1: 1). e supernatant was analyzed by UPLC-Q-TOF-MS/MS. e blank urine sample was processed in the same way.

Metabolism in Rat Liver
Microsomes. e total volume of the incubation system is 500 μL, including phosphate buffer (0.1 M, pH 7.4) 470 μL, and NADPH generating system . e process is completed on an ice bath. Marein blank control group: Take the above incubation system, add 5 μL of aqueous solution to an ice bath, incubate at 37°C for 5 min, and then add 15 μL of liver microsomes to the ice bath. After gently mixing the incubation system by hand, start the incubation at 37°C in a water bath. e incubation time is 0, 5, 15, 30, 45, 60, 90, and 120 min, and each sample is paralleled 3 times. When the reaction is complete, stop the reaction with the same volume of ice acetonitrile to completely precipitate the protein, centrifuge at 4,200 g for 10 min, aspirate the supernatant and centrifuge at 19,500 g for 10 min, and inject 2 μL for analysis.
Marein inactivated liver microsomes group: First inactivate liver microsomes, take the incubation system, add 5 μL of the marein reference substance stock solution on an ice bath, incubate at 37°C for 5 min, and then add 15 μL of inactivation to the ice bath. e following procedures were similar to the blank control group.
Marein liver microsomes group: Take the incubation system, add 5 μL of the marein reference substance stock solution to an ice bath, incubate at 37°C for 5 min, and then add 15 μL of liver microsomes to the ice bath. e following operation was the same as the blank control group.

UPLC-MS/MS Conditions.
Used UPLC-Q-TOF-MS/MS 9030 system (Shimadzu, Japan), chromatography column: Shim-pack GISS C18 column (100 × 2.1 mm, 1.9 μm). Mobile phase: A phase 0.1% formic acid water, B phase acetonitrile, gradient elution, elution gradient: 0∼1 min, 5∼10% B; MS operation conditions were as follows. e Q-TOF-MS spectrometer was configured with an electrospray ion source (ESI) operating in the negative and positive ion modes. Ion source interface voltage is − 3.0 Kv. Nitrogen is used as the drying gas and the atomizing gas. e drying gas flow rate is set to 10 L/min, and the atomizing gas flow rate is set to 3.0 L/min. Air is used as heating gas, and the flow rate is set to 10 L/min; argon is used as collision gas; desolvent  Evidence-Based Complementary and Alternative Medicine tube temperature is 250°C; heating block temperature is 400°C; and interface temperature is 300°C. Scan modes are MS Scan (m/z 100-500; 500-1,000) and MS/MS (m/z 50-500; 50-1,000). e collision energy (CE) is 35 ± 17 V.
In the study of metabolism in vivo, the injection volume was 5 μL, while that in vitro metabolism was 2 μL.

Data Analysis.
e data obtained by UPLC-Q-TOF-MS/MS were analyzed with Formula Predictor Server software. Target fragments are analyzed by ACD/Labs software accurate mass calculator (Shimadzu City, Japan).
In the liver microsomes study, the 0 min peak area of marein was regarded as 100%, and other time points were compared with the 0 min peak area to obtain the remaining percentage (area%). According to the natural logarithm of area% and incubation, the time slope k was obtained by linear regression. According to formula (1), the half-life t 1/2 / min of marein in liver microsomes could be obtained. According to formulas (2)-(5), the half-lives in liver microsomes, intrinsic clearance rate CL int , in vivo intrinsic clearance rate CL′ int , liver clearance rate CL H , and liver extraction rate ER were calculated.
e physical and chemical parameters: e liver tissue per kilogram of body weight for rat (Liver (g)/BW (kg)) was 40 g. Q is hepatic blood flow (66 ml/min/kg for rat), and CL′ int is the intrinsic clearance. e microsomal protein per gram of liver tissue (microsomes (mg)/liver (g)) was 45 mg.

Network Pharmacology.
e Mol2 structural formulas of marein and its metabolites were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and then imported into the PharmMapper database (http://www.lilabecust.cn/pharmmapper/) to predict the possible targets of the ingredients [27][28][29], screened with a normalized fit score greater than 0.5, and then standardized through the Uniprot database (https://www.uniprot.org/) [30]. Select this species as "Homo sapiens" and get multiple targets for each chemical component to exert pharmacological effects. e intersection of targets was processed; duplicate values were deleted; and the PPI protein interaction network was constructed by importing the STRING website (https://www.string-db.org/) [31]. Kernel targets were screened by importing Cytoscape software [32]. After that, KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathway enrichment and GO (Gene Ontology) analysis were carried out in the Database for Annotation, Visualization, and Integrated Discovery (DAVID; https://david.ncifcrf.gov/summary.jsp) [33]. e major action pathways were screened out to explore the relationship between "component-important target-pathway."

Identification of Marein Metabolites in Rats after Oral
Administration. UPLC-Q-TOF-MS/MS was used to study the chromatographic and mass spectrometric cleavage behavior of marein and its metabolites.
irty metabolic components for oral administration were identified, and 25 and 19 metabolites were detected in plasma and urine separately. e detailed metabolites information after orally administrated of marein was summarized in Table 1, and extracted ion chromatograms of metabolites were shown in Figure 1. e results show that no prototype components were found in the rats' plasma and urine after the rats were given marein by gavage. It means that after intragastric administration, marein is metabolized and mainly exists in the form of metabolites in the blood.   Evidence-Based Complementary and Alternative Medicine the product ions at 151 and 135 of marein. Combined with the molecular formula, it is speculated that it has 3 more hydroxyl groups. erefore, it is preliminarily determined to be a dehydrogenation and hydroxylation metabolite. e retention time of metabolite M2 is 6.37 min. e anion first-order mass spectrometry shows that the excimer ion peak [M-H] − is m/z 495.0708, and its molecular formula is predicted to be C 21   is predicted to be C 21 H 20 O 13 , which is an isomer with M3. e MS mass spectrum correspondingly produces characteristic fragment ions m/z 151.0033 and 135.0438. It is also the dehydrogenation and hydroxylation metabolites of marein.     Figure 6: e metabolic pathways of marein after injection of plasma and urine.

Identification of Marein Metabolites in Rats after
Injection.
e prototype compounds and metabolites in the urine and blood samples of rats after the tail vein injection of marein were analyzed. By comparing the retention time in the chromatograms of the administration group and the blank group to track the metabolites, 31 metabolites were detected in injection, and 23 and 26 were detected in plasma and urine separately.
Among them, M′0 is the prototype marein. And M′1 has the same molecular weight as M′0, which may be presumed to be the hydrogenation product after dehydrogenation. M′2 and M′3 can be inferred to be hydroxylated and sulfated metabolites according to their molecular weight and molecular formula. M′4 and M′5 have a molecular weight of 80 more than that of M′0. M′6 and M′7 are the hydroxylated products after the dehydrogenation of 2 hydrogens. Compared with M′0, the molecular weight of M′8, M′9, and M′10 are 2 less, which is supposed to be the removal of 2 hydrogens. e molecular weight of M′11, M′12, and M′13 are 16 less, so they may be dehydroxylated products. M′14-M′16 are 14 more than M′0 and are methylated products. M′17 is inferred to be the removal of 2 hydroxy groups after the addition of 2 hydrogens. M′18-M′21 is presumed to be a glycosylation metabolite, and M′22 is presumed to be a deglycosylation product and an aglycone of marein. M′23-M′27 may be the methylation products of M′6-M′7. And M′28-M′31 may be the sulfuration products of M′14-M′16. e detailed metabolites information after injection of marein was summarized in Table 2, and extracted ion chromatograms of metabolites were shown in Figure 3.
More interestingly, the prototype components were detected after the tail vein injection, and isomerization also occurred, that is, the two isomers marein and flavanomarein. e structure was shown in Figure 4. After intravenous administration, the two transformed into each other. It provides a reference for further research on the pharmacological studies of marein (chalcone glycoside) and flavanomarein (dihydroflavonoid glycoside). Regarding the isomerization reaction, it has also been reported in the metabolism of flavonoids with similar structures: rat gavage liquiritigenin (LG; dihydroflavonoids), isoliquiritigenin (ILG; chalcone), liquiritin (LQ; dihydroflavonoid glycoside), and isoliquiritin (ILQ; chalcone glycoside). Its chalcone components could realize the conversion with the corresponding dihydroflavonoids [34].

Oral Administration and Injection Metabolic Pathways
Analysis.
e possible metabolic pathways of marein via oral administration and injection were shown in Figures 5  and 6, respectively. e metabolic pathways include sulfation, hydrogenation, hydroxylation, methylation, glucuronidation, hydrolysis, dehydroxylation, isomerization, and dehydrogenation.
Marein belongs to chalcone glycoside. According to the results of oral and injection studies, it can be hydrolyzed into its aglycones in vivo, and its aglycone M25 (chalcones) could be further metabolized. Studies on the metabolism of components with similar structures have been reported. For example, after intragastric administration of isoliquiritin (chalcone glycoside) and isobavachalcone (chalcones) in rats, metabolic pathways in vivo include glucuronidation, hydrolysis, isomerization, sulfation, hydrogenation, hydroxylation, and other metabolic reactions [35]. e metabolic rule of marein is consistent with that of other chalcone glycosides, indicating the common rule of chalcone glycoside metabolism.
By comparing the experimental results of oral and injection of marein, it is found that their metabolic pathways include hydroxylation, glucuronidation, methylation, hydrolysis, and dehydrogenation. e difference is that after oral administration, marein was not detected in the original   form, and mainly existed in the form of metabolites in blood. While injection administration, that is, in the absence of gastrointestinal absorption and liver first-pass effect, the prototype component of the glycoside can be detected in the body. It can be seen that the first-pass effect of the gastrointestinal tract and the liver plays an important role in the oral metabolism of marein, which makes the metabolites different between the two administration methods.

Metabolism of Marein in Rat Liver
Microsomes. Liver microsomes can effectively evaluate the influence of metabolic enzymes on compound metabolism. In this experiment, an in vitro incubation system for rat liver microsomes was established. e UPLC-Q-TOF-MS/MS method was used to identify the metabolites of marein in liver microsomes and to study the metabolic stability in vitro. e data of Ln (area%) and the time of incubation were shown in Figure 7; the data of area%, Ln (area%), and the time of incubation were shown in Table 3; and the data of the halftime period, CL int , CL′ int , CL H , and ER were shown in Table 4. e results show that the in vitro metabolic half-life t 1/2 of marein in liver microsomes is 177.69 min and the liver extraction rate is 0.15. It shows that the metabolic stability of marein in liver microsomes is good, indicating that liver microsomes have no significant effect on the metabolism of marein. Since the prototype components of marein were not detected in the body after intragastric administration, we speculate that liver metabolism is not the main reason for the low bioavailability of marein. According to existing research, the gastrointestinal tract may play a key role in the metabolism of marein. e study of rutin gives us some enlightenment. Rutin can be combined with enzymes in the intestine after oral administration. e enzymes in the intestine hydrolyze most of the rutin into quercetin, and only a small part of the rutin can be absorbed into the blood. Given the similar structure of marein, flavanomarein, and rutin, it is speculated that after oral administration of marein in rats; the enzymes in the intestinal tract will hydrolyze marein; and its metabolites are absorbed into the blood.

Network and Target Biological Function Analysis.
Predictive analysis of the related targets is based on marein and its metabolites: okanin (marein's aglycon, M′1) and flavanomarein (the isomer of marein, M24 or M′22). Two hundred and sixty-five targets were obtained. en input the obtained targets into STRING to predict the interaction relationship between protein and protein. e species was selected as "Homo sapiens," and set a score greater than 0.700 as the basis for high confidence in the interaction, hiding the disconnected nodes. Export the tsv format file and import it into Cytoscape 3.6.1 for network visualization and topological attributes analysis. e higher the degrees of nodes are, the more important the nodes are to maintain the stability of the entire network. We sorted the degree values and selected the core targets with the corresponding degree values greater than four times the median value as the potential key targets related to activity and visualized them (Figure 8(c)). e nodes with higher degrees are in a brighter color, and the node size and edge size represent the betweenness centrality and edge betweenness of the nodes, respectively. e top 5 core targets are SRC, AKT1, EGFR, HRAS, and HSP90AA1. KEGG signaling pathway enrichment and GO function analysis were performed on 16 targets with degree values greater than quadruple the median as the core targets by using the Functional Annotation tool in the DAVID database.
In Figure 8(a), the KEGG pathways were sorted by their nominal count values, mainly involving proteoglycans in cancer, pathways in cancer, estrogen signaling pathway, and other pathways. e most enriched pathway is proteoglycans in cancer, which contains 10 genes, followed by pathways in cancer, which contains 10 genes, and the estrogen signaling pathway contains 8 genes. According to the publications, the 3 components [36,37] may play an important role in antitumor and estrogen signaling pathways.
In the GO function analysis, a total of 177 biological processes were enriched. e results showed that the key targets are closely related to the regulation of biological functions such as the ERBB2 signaling pathway, positive regulation of nitric oxide biosynthetic process, phosphatidylinositol-mediated signaling, and protein autophosphorylation. Regarding biological processes, a total of 8 processes were enriched with P < 0.01 and count ≥5, which were mainly related to signal transduction, negative regulation of the apoptotic process, cell proliferation, and positive regulation of transcription from RNA polymerase II promoter. In cell components, it was mostly related to the nucleus, cytosol, plasma membrane, and cytoplasm. Among the molecular function, P < 0.01 and count ≥5 were enriched in a total of 4 biological processes, which were related to protein binding and ATP binding, enzyme binding, and identical protein binding. See Figure 8(b) for details.
A network relationship diagram of "components-core targets-important pathways" using Cytoscape software visualization analysis was constructed. As shown in Figure 8(d), this network contains 31 nodes (3 chemical components, 15 target points, and 13 related pathways) and 115 edges (the relationships between them). e larger the point in the figure is, the more important the position in the network is. Marein, flavanomarein, and okanin mainly acted on 15 targets such as EGFR, AKT1, SRC, HRAS, and CASP3; the detailed related topology information is shown in Table 5. e targets involve proteoglycans in cancer, pathways in cancer, estrogen signaling pathway, Ras signaling pathway, and others. e targets exert anti-tumor and anti-inflammatory effects, which are consistent with pharmacological research on the activity of marein.
Marein and its metabolites may act on the same target to exert a similar activity. e intersection of the core targets of the three components shows that they may act on the six common targets GSTP1, PTPN1, SRC, ME2, HMGCR, and CASP3, and SRC are the core targets in the whole network. SRC; a proto-oncogene that is a member of the protein tyrosine kinase family, is one of the key factors leading to the increase of malignancy in many tumors [38].

Conclusion
A rapid and reliable UPLC-Q-TOF-MS/MS method was established for the first time to identify the metabolites of marein in vivo. e results remind us that when studying the efficacy and toxicity of flavonoids, the actual form of the flavonoids in vivo must be considered. e componenttarget-pathway network of marein, its aglycones, and flavanomarein was further established to illustrate their activity, and we found that marein and its metabolites act on key targets of EGFR, AKT1, SRC, HRAS, CASP3, and so on. It mainly involves pathways such as proteoglycans in cancer, pathways in cancer, estrogen signaling pathway, and Ras signaling pathway. e study provides a scientific basis for future research on the route of administration of flavonoids, the mechanism of action, and the discovery of lead compounds from active metabolites.