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Filtration of Active Components with Antioxidant Activity Based on the Differing Antioxidant Abilities of Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus through UPLC/MS Coupling with Network Pharmacology

Filtration of Active Components with Antioxidant Activity Based on the Differing Antioxidant... Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 5547976, 13 pages https://doi.org/10.1155/2021/5547976 Research Article Filtration of Active Components with Antioxidant Activity Based on the Differing Antioxidant Abilities of Schisandrae Sphenantherae Fructus and Schisandrae Chinensis FructusthroughUPLC/MSCouplingwithNetworkPharmacology 1,2,3 1 1 4 Yang Xin , Yang Yang, Kaichen Yu, and Haijun Wang College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar 161006, China Heilongjiang Academy of Chinese Medical Sciences, Harbin 150036, China Heilongjiang Provincial Key Laboratory of Catalytic Synthesis for Fine Chemicals, Qiqihar University, Qiqihar 161006, China School of General Medicine and Continuing Education, Qiqihar Medical University, No. 333, Bukui Street, Qiqihar 161006, China Correspondence should be addressed to Yang Xin; cc.xinyang@163.com and Haijun Wang; whjxy0802@163.com Received 19 January 2021; Revised 20 June 2021; Accepted 7 July 2021; Published 22 July 2021 Academic Editor: Hicham Harhar Copyright © 2021 Yang Xin et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. -is study attempted to filter active components with antioxidant activities based on the differing antioxidant abilities of Schisandrae Sphenantherae Fructus (SSF) and Schisandrae Chinensis Fructus (SCF). First, the antioxidant activity of SSF and SCF was evaluated through the DPPH free radical scavenging method and compared with the half maximal inhibitory concentration (IC ) value. Next, components of SSF and SCF were detected by employing ultrahigh-performance liquid chromatography-Q- Exactive Orbitrap mass spectrometry (UPLC-QEO/MS) technology, and differential compounds were screened out as potential antioxidant compounds by using Compound Discover 3.1 Software. After that step, in order to verify the antioxidant compounds, the network method was applied. Biological targets were searched in the GeneCards database, and that related to antioxidant ability were selected in the Comparative Toxicogenomics Database (CTD). Finally, the pharmacology network was constructed. Results showed that SSF and SCF possessed different compounds and antioxidant abilities. A total of 14 differential compounds such as c-schizandrin, schisandrin B, schisandrin, and tigloylgomisin H between them were screened out and identified. Twenty targets associated with antioxidant activity contained MAP2K1, MAPK8, RPS6KB1, PRKCB, HIF1A, and so on were investigated. -irty-six pathways contained HIF-1 signaling pathways, choline metabolism in cancer, serotonergic synapse, Fc epsilon RI signaling pathway, GnRH signaling pathway, and so on related to the above twenty targets were identified. -e pharmacology network analysis indicated that the differential components may be helpful in treating various diseases, especially cancer, by exerting antioxidant activity. In conclusion, this study provided a novel method for identifying active components with anti- oxidant activity in SSF and SCF, and this method may be applicable for the filtration of bioactive components in other herbs. Free radicals are constantly produced in the body due to 1.Introduction exposure to the environment through processes such as Antioxidation is a process that can effectively inhibit the respiration (oxidation reaction), air pollution, and radiation. oxidation reaction caused by free radicals. -e mechanism of Free hydroxyl radicals are associated with aging stress [1], antioxidation involves either an action on free radicals or the autoimmunity [2], bone loss, and cancer development [3]. consumption of substances that can easily generate free However, substances that possess antioxidant activity can radicals. terminate or inhibit the oxidation process by scavenging free 2 Evidence-Based Complementary and Alternative Medicine radicals [4]. -us, maintaining the balance of free radicals (Alabama, USA), and L-ascorbic acid (vitamin C (VC)) was and the antioxidant active substance in the body can delay purchased from InnoCHEM (InnoCHEM, China). Ana- aging. -erefore, finding antioxidant active components is of lytical-grade anhydrous ethanol was used (Tianli, China). significance for developing antioxidant health products and pharmaceuticals to cure diseases caused by oxidative 2.2. Sample Preparation. SCF and SSF were prepared using damage, as well as for studying the mechanisms governing the reflux extraction process [11]; the extraction method for antioxidant activity. them was optimized based on the DPPH radical clearance Schisandrae Sphenantherae Fructus (SSF) and Schisan- rate by utilizing an L9(3 ) orthogonal design. During or- drae Chinensis Fructus (SCF) are both well-known Chinese thogonal experiments, factors concerning extract times, herbal medicines, and SSF is primarily produced in the reflux time, and material-liquid ratio were all inspected with central and southern part of China, whereasSCF is primarily four levels. -e optimal extraction process was as follows: produced in northern China. Although these medicines first, SCF or SSF was weighed 2.5g. Next, the herbal med- possess some similar pharmacological actions, such as an- icine was mixed with 20 volumes of water and then boiled for tioxidant, anti-inflammatory, and anxiolytic effects [5], SCF 2h, 5 times. -e extract was then filtered, mixed, and and SSF exhibit differences in chemical components [6] and concentrated to 50mL. After that, 2mL of the extract was other pharmacological actions [7], which might be the measured and dried. Finally, the residue was dissolved using reason why SCF is more popular in Chinese medicine than 2mL anhydrous ethanol before detection. SSF. -erefore, conducting a study simultaneously com- paring the components and biological activities, as well as their connections, was of interest. 2.3.DPPHRadicalClearanceTest. DPPH was dissolved with Regarding Chinese herbal medicine, because of the 95% ethanol aqueous solution to prepare 0.1mmol/L of multiple components of these medicines, the interactions DPPH solution and 2mL of it was mixed with 2mL sample. between them and treated organisms is very complex, which -e mixture was then detected at 517nm using a UV makes studies on the mechanisms governing the medicines spectrometer after 30min. difficult to conduct. Fortunately, the concept of “network pharmacology” was proposed by Hopkins in 2008 [8] and has been widely employed in research on Chinese herbal 2.4. Condition of Detection. -e absorbance of the SCF and medicine [9]. With the help of network pharmacology, the SSF extracts was measured on an ultraviolet-visible spec- correlation between drugs and action targets could be trophotometer TU1901 (Persee Co., China) in the spectral predicted through computer virtual computation, network scan mode in the range of 510∼516nm. database retrieval, network modeling, and network analysis. Compounds inSCF andSSF were detected on UPLC-MS Compared to traditional pharmacology, network pharma- equipment consisting of a Dionex UltiMate 3000 UHPLC cology exhibits the features of “multitarget, multieffect, and and Q-Exactive Orbitrap MS with an electrospray source in complex disease” [10]. Based on the above characteristics, positive-ion mode (-ermo Co., USA). network pharmacology has been recognized by an increasing UPLC separation was performed on a TM C18 column number of scholars, and network pharmacology has been (2.1 ×50mm, 2.6μm, -ermo Co., USA). Formic acid was increasingly utilized to study pharmacological effects. added to the mobile phase, which consisted of methanol (A) In this study, a network pharmacology method was and 0.1% formate water (B), to improve ionization efficacy employed to verify the antioxidant active compounds fil- and acquire a better peak shape. -e flow rate was set at −1 tered by comparing the components in SCF with those of 0.3mL·min . UPLC resolution was optimized with gradient SSF based on the results of UPLC/MS technology. Specifi- elution as follows: 0∼2min, 40%∼60% A; 2∼6min, 60%∼ cally, this report presents a novel method for determining 100% A; and 6∼8 min, 100% A. -e column temperature was the antioxidant components of these medicines, which 25 C, and the injection volume was 0.5µL. depends on connecting the differing antioxidant abilities of -e optimal mass spectrometry signal was obtained as SCF and SSF with the differential components between follows: capillary (+4.0kV), desolvation temperature them, as well as verifying these results through the strategy of (350 C), S-lens voltage (50V), shield gas (35arb), aux gas network pharmacology. -e method described in this study (10arb), scan type (full scan), and scan range (100∼1200Da). may facilitate the identification of antioxidant compounds in -e collision energy for MS was set at 10, 30, and 40eV to other herbal medicines. acquire abundant fragment ions. -e UV method was confirmed by inspecting the ac- curacy and stability of the absorbance of the same SCF 2.Experiment sample six times. -e stability was determined by testing six 2.1. Solvent and Medicine. Schisandrae Chinensis Fructus times in 30min. A relative standard deviation percentage (Schisandra chinensis (Turcz.) Baill., fruit, dried) was pur- (RSD%) of absorbance below 1% was considered to indicate chased from Qi Tai Pharmacy (Qiqihar, China). Schisandrae a good detection method. Sphenantherae Fructus (Schisandra sphenanthera Rehd. et -e UPLC-MS method was validated by inspecting the Wils., fruit, dried) was purchased from Xing Kang Pharmacy accuracy and stability through the detection of six com- (Qiqihar, China). -e reference standard for 1,1-diphenyl-2- pounds in both SCF and SSF. -e accuracy was determined picrylhydrazyl (DPPH) was purchased from Avanti by injecting the same SCF sample continuously six times. Evidence-Based Complementary and Alternative Medicine 3 -e stability was implemented by injecting the same SCF the accuracy and stability of the analytical method were sample at 0, 12, 24, 32, 40, and 48h. -e RSD% of retention acceptable. time and peak area below 5% was considered to indicate a good detection method. 3.2. Filtration of the Optimal Extraction Process for Antioxi- dantActivity. -e results of the orthogonal test are shown in Table 1. It can be seen that, through the optimization of 2.5.DataAnalysis. -e UV absorbance data of SSF and SCF the extract method, sample prepared by the extraction were used to calculate the DPPH radical clearance, as well as process with the material-liquid ratio of 1 : 20, extracted the IC value. First, the DPPH radical clearance was cal- for 5 times and 2.0 h/time showed the optimal DPPH culated as (Abs −Abs )/Abs ×100%. Second, the blank sample blank radical clearance rate, which is used for the extraction of IC value was acquired through curve fitting, with extract SSF and SCF. concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0mg/mL serving as the X-axis and the DPPH radical clearance value serving as the Y-axis, by GraphPad software. -ird, the IC values of 3.3.EstimationofAntioxidantActivitybetweenSSFandSCF. the medicines were compared with that of vitamin C to -e DPPH radical clearance ability of SSF and SCF was estimate their antioxidant ability. estimated by comparing the IC value with vitamin C (VC); All UPLC/MS raw data were acquired by Q-Exactive the lower the IC value was, the better the antioxidant Tune (-ermo Co., USA) and processed with Compound activity was. -e results showed that the IC values of SCF, Discoverer 3.1 (-ermo Co., USA). Next, a data list con- SSF, and VC were 0.4518±0.0008mg/mL, 0.4928± taining data such as molecular weight, retention time, peak 0.0015mg/mL, and 0.2225±0.0037mg/mL, respectively, intensity, and p which indicated thatSCF exhibited better antioxidant ability value of SCF versus SSF was acquired. Compounds with than SSF. a p value<0.05 were considered to be significantly different between SCF and SSF. Principal component analysis (PCA) 3.4.FilterationandIdentificationofthePotentialAntioxidant was used to observe the overall classification of the data. Active Components. To determine the reason for the anti- Next, the differential compounds were identified by oxidant ability of SSF being better than that of SCF, it was searching the mzCloud, ChemSpider, PubChem, NIST surmised that there were some compounds that showed a Chemistry WebBook databases, and references based on higher intensity inSCF than inSSF; therefore, the differential their MS spectra. compounds between SSF and SCF were filtered. -e targets of differential compounds were screened out First, samples of SSF and SCF were detected by UPLC/ in the Swiss Target Prediction database after the canonical MS, and total chromatograms were obtained using SMILES number was obtained from PubChem and com- Q-Exactive Tune (-ermo Co.). -e clear difference in the pounds were standardized by the UniProt database. -e peak amount and intensity between SSF and SCF is depicted CTD and GeneCards databases were searched for the targets in Figure 1. of antioxidant function. After that step, the common targets Second, the overall difference between SSF and SCF was of differential compounds and antioxidant function were observed through unsupervised principal component imported into the STRING database, and “multiple pro- analysis (PCA) (Figure 2), and variables making significant teins” and “human sources” were checked. Next, protein- contributions to discrimination between them were selected protein interactions (PPIs) were depicted using Cytoscape. based on a p value<0.05 by using Compound Discoverer 3.1 Next, Gene Ontology (GO) and Encyclopedia of Genes and software. Next, the Traditional Chinese Medicine Systems Genomes (KEGG) enrichment analyses were performed Pharmacology Database (TCMSP, https://tcmspw.com/ using the Metascape database. Finally, a network pharma- tcmsp.php), mzCloud (https://www.mzcloud.org/), Chem- cology diagram was drawn with the help of Cytoscape Spider (https://www.chemspider.com/), PubChem (https:// software. pubchem.ncbi.nlm.nih.gov/), and the National Institute of Standards and Technology (NIST) Chemistry WebBook (https://webbook.nist.gov/chemistry/) were searched to de- 3.Results termine the molecular weight and the molecular formula of different variables, and their MS 3.1. Evaluation of Methodology. Both of the methodologies spectra were compared with for UV and UPLC/MS were performed. For UV, the RSD% those in references to identify the chemical structures. A total of accuracy and stability was less than 0.5%. Regarding the of 14 compounds were identified, and all their intensity ratios UPLC/MS method, the RSD% accuracies for schisandrin, in SCF versus SSF were greater than 1; therefore, they were gomisin J, schisantherin B, angeloylgomisin P, schilancifo- initially considered antioxidant compounds, as shown in lignan A, and schisantherin C were 0.97%, 1.64%, 1.84%, Table 2. 1.72%, 0.85%, and 2.04%, respectively, and the RSD% of Among these compounds, espatulenol was proposed by stability for them in SSF was 2.55%, 2.44%, 2.90%, 0.68%, comparing the MS spectrum with that in the NIST 2.98%, and 3.52%, respectively, whereas those in SCF were Chemistry WebBook database, c-schizandrin, schisantherin 3.36%, 3.60%, 0.45%, 3.24%, 4.95%, and 0.91%, respectively. B, schisantherin C, tigloylgomisin H, gomisin J, ange- All the RSD% values were less than 5%, which indicated that loylgomisin P, angeloylgomisin H, gomisin M1, gomisin E, 4 Evidence-Based Complementary and Alternative Medicine Table 1: L9(3 ) orthogonal array design matrix and experimental 3.6. Protein-Protein Network Construction. -e protein- results. protein interaction (PPI) network diagram was constructed using Cytoscape software (Figure 5). Figure 5 shows that the No. A B (g/mL) C (/h) D DPPH clearance% network graph contained targets and 24 edges. Each target 1 3 (1) 1 :08 (1) 1.5 (1) 1 22.81 connected several functions. -e larger the degree value was, 2 3 (1) 1:10 (2) 2.0 (2) 2 27.34 the larger the node was, and the larger the combined score 3 3 (1) 1:20 (3) 2.5 (3) 3 27.50 value was, the thicker the edge was. Among these genes, the 4 4 (2) 1 :08 (1) 2.0 (2) 3 26.88 top 3 genes with larger nodes included MAPK8, CCND1, 5 4 (2) 1:10 (2) 2.5 (3) 1 26.72 6 4 (2) 1:20 (3) 1.5 (1) 2 30.63 and RPS6KB1, which indicated that they were important 7 5 (3) 1 :08 (1) 2.5 (3) 2 30.47 targets on which the potential antioxidant components 8 5 (3) 1:10 (2) 1.5 (1) 3 30.16 acted. 9 5 (3) 1:20 (3) 2.0 (2) 1 41.39 K1 25.883 26.720 27.867 30.307 — K2 28.077 28.073 31.870 29.480 — 3.7. Gene Ontology Enrichment Analysis for Targets. GO K3 34.007 33.173 28.230 28.180 — (gene ontology) enrichment analysis of potential targets was R 8.124 6.453 4.003 2.127 — performed using the Metascape database, and the functions Note: A: reflow times; B: material-liquid ratio; C: reflow time; D: blank. were enriched under the conditions of p value <0.01 and enrichment factor >1.5. Figure 6 shows the top 20 most significant functions. In Figure 7, the point and gomisin K1 were proposed by comparing the MS size represents the number of genes with the same function; spectrum with that in references [12–17], and schilancifo- the larger the point size is, the more genes there are. -e lignan A was speculated based on its MS fragments [18]. results showed that potential antioxidant compounds af- Schisandrin and schisandrin B were verified by their stan- fected numerous biological functions involving biological dards. -e ion of m/z 455.2040, which was one significantly processes, cellular components, and molecular functions, different compound betweenSSF andSCF, was selected as an such as the regulation of aging, the active regulation of example to illustrate the compound identification process. programmed cell death, the positive regulation of reactive First, the extract ion spectrum of m/z 455.2040 was acquired oxygen metabolism, the regulation of neuronal apoptosis, at t 3.71min. -en, its MS spectrum was acquired under positive regulation of the cell cycle, and regulation of the 40eV, which is shown in Figure 3. Figure 3 shows that collagen metabolism process. -ese targets might slow the fragment ions ofm/z 415.2113, 400.1872, 384.1931, 369.1696, occurrence and development of aging and hypoxia caused by 359.1479, and 353.1750 exerted the same fragmentation with the oxidation process by participating in the process of that of schisandrin reported in references, corresponded to + + + antioxidant reactions. [M+H-H O] , [M+H-H O-CH ] , [M+H-H O-OCH ] , 2 2 3 2 3 + + [M+H-H O-OCH -CH ] , [M+H-H O-C H ] , and 2 3 3 2 4 8 [M+H-H O-OCH -OCH ] based on high-resolution mass 2 3 3 3.8. Pathway Analysis for Targets. -e top 36 enriched spectrometry. Hence, it was deduced to schisandrin initially. pathways of the above 20 targets were obtained from the Further, the schisandrin standard was detected by the same DAVID (Database for Annotation, Visualization, and In- UPLC/MS condition for verifying the deduction, which tegrated Discovery) database, which is shown in Figure 7. showed that their retention time and MS spectrum were -e length of the column represents the number of genes matched completely. -erefore, the ion of 455.2040 was participating in the pathway; the longer the column is, the identified as schisandrin. -e proposed fragmentation more genes there are. Among these pathways, pathways in pathways of schisandrin are shown in Figure 4. cancer, the HIF-1 signaling pathway, insulin resistance, the insulin signaling pathway, hepatitis B, the AMPK signaling pathway, the MAPK signaling pathway, the TNF signaling 3.5. Prediction of the Targets of Antioxidant Compounds. pathway, and the FoxO signaling pathway were reported to A total of 441 targets on which the abovementioned potential be related to antioxidant activity [19–27]. antioxidant compounds acted were obtained. At the same time, 4151 targets associated with antioxidant activity were obtained by searching the GeneCards database, and 3946 of 3.9. Construction of Compound-Target-Pathway Network. them were retained after UniProt processing. Furthermore, -e compound-target-pathway network was constructed by 4531 targets related to antioxidant activity were obtained by connecting 14 differential compounds, 20 targets, and 36 searching the CTD database, and 150 of them were retained pathways. Figure 8 shows that there were a total of 70 nodes after UniProt processing. -ere were 147 common targets in and 230 edges. -e larger the node was, the more nodes were the GeneCards database and CTD database. Furthermore, connected to it. -erefore, angeloylgomisin H, aigloylgo- the 147 targets were compared to the abovementioned 441 misin H, angeloylgomisin P, schisantherin C, and schisan- targets, and it was found that 20 targets on which the an- therin B were deemed to have stronger antioxidant activity tioxidant compounds acted were related to antioxidant than other compounds. MAP2K1, MAPK8, PRKCB, and activity; these targets are listed in Table 3. CCND1 were considered to be the primary antioxidant effect Evidence-Based Complementary and Alternative Medicine 5 5.08 0.47 90 4.38 3.51 6.49 4.49 6.82 4.07 9.20 7.10 5.01 5.68 0.66 2.06 3.38 1.91 3.15 9.09 9.40 0.84 6.40 9.58 1.25 2.71 10.10 7.79 8.37 0 123456 7 8 9 10 11 Time (min) (a) 8.72 1.09 7.30 7.78 2.17 0.63 10 10.16 9.06 9.66 3.44 9.47 3.14 3.37 6.20 1.92 2.07 9.10 5.67 0.46 0.48 9.12 4.60 6.80 4.49 9.19 9.23 7.10 6.48 5.08 4.35 (b) Figure 1: Mirror image of the total ion chromatogram for SCF (a) and SSF (b). effects, the main effects that were essential for quality dif- targets. Pathways in cancer, choline metabolism in cancer, and proteoglycans were considered to be the primary ferences were not clear. -e antioxidant activity or com- pathways involved in antioxidant processes. ponents of medicines have long been compared by medicinal scholars [28, 29], but to the best of our knowledge, studies on the relationship of antioxidant ability and differential 4.Discussion components have not been performed to date. -erefore, this study described their relationship and predicted the SSF and SCF, which serve as typical “medicine-food ho- mology” herbal medicines, showed similar pharmacological functional targets of antioxidant activities using a network pharmacology strategy. -e compound-target-pathway effects, such as antioxidant activity. Nevertheless, SCF was generally more popular thanSSF because it was said thatSCF network diagram is shown in Figure 8, in which there were a total of 70 nodes and 230 edges. -e larger the degree value possessed higher quality than SSF. Regarding SSF and SCF, was, the larger the nodes were, and the stronger the because both of these medicines had multipharmacological 6 Evidence-Based Complementary and Alternative Medicine –10 –20 –60 –40 –20 0 20 40 PC 1 (44.5%) SSF SCF Figure 2: PCA for samples of SCF and SSF. Table 2: Components of potential antioxidant activities. Deduced t Elemental Ion Calculated Deviation No. MS compound (min) composition adduction mass (ppm) b,d + 1 c-Schizandrin 2.76 C H O [M+H] 401.1959 −0.5 386.1723, 370.1768, 359.1487, 355.1532, 337.1432 23 28 6 203.1795, 193.1587, 175.1481, 163.1481, 147.1168, b,d + 2 Espatulenol 3.40 C H O [M+H] 221.1900 −1.4 15 24 133.1012, 107.0858, 95.086 455.2032, 415.2113, 400.1872, 384.1931, 369.1696, a,b + 3 Schisandrin 3.71 C H O [M+Na] 455.2040 −2.0 24 32 7 359.1479, 353.1750 b,d + 4 Tigloylgomisin H 3.91 C H O [M+Na] 523.2302 −0.6 493.1786, 455.2077, 423.1418, 401.1621, 383.1506 28 36 8 [M+Na] 411.1778 −1.5 5 Gomisin J 3.99 C H O 357.1721, 325.1453, 319.1181, 297.1483, 287.0919 22 28 6 [M+H]+ 389.1943 −3.9 Schisantherin B or 6 4.09 C H O [M+Na] 537.2095 −0.7 437.1573,415.1750,371.1498,356.1246 b 28 34 9 angeloylgomisin P Angeloylgomisin P 7 4.12 C H O [M+H] 537.2095 −1.5 437.1573,415.1750,371.1498,356.1246 b 28 34 9 or schisantherin B Angeloylgomisin 416.1823, 387.1798, 372.1564, 356.1614, 342.1453, 8 4.13 C H O [M+Na] 523.2302 −0.6 b,d 28 36 8 H 326.1503 Schilancifolignan 9 4.25 C H O [M+H] 431.2064 −1.2 493.1840,455.2039,423.1422,383.1489 c,d 24 30 7 b + 10 Schisantherin C 4.50 C H O [M+H] 515.2275 −1.4 385.1649, 355.1543, 343.1183, 316.0944 28 34 9 b,d + 11 Gomisin K1 4.65 C H O [M+Na] 425.1934 −0.7 410.1701, 395.1464, 379.1516 23 30 6 b + 12 Gomisin E 4.97 C H O [M+H] 515.2275 −0.2 469.2206, 385.1646, 355.1538, 343.1179, 316.0941 28 34 9 b,d + 13 Gomisin M1 5.07 C H O [M+Na] 409.1621 −1.0 394.1393, 363.1185, 333.1724 22 26 6 a,b,d + 14 Schisandrin B 5.37 C H O [M+H] 401.1959 −0.7 300.0990,270.0883,242.0936,331.1174,386.1723,401.1953 23 28 6 -e superscript “a” represents compounds identified by standard, superscript “b” represents compounds identified by literature reports, superscript “c” represents compounds speculated based on its MS fragments, and superscript “d” represents compounds solely found in SCF. antioxidant activity of the compounds was. Among the 14 namely, MAP2K1, MAPK8, PRKCB, and CCND1, showed differential compounds, seven compounds, namely, larger nodes than the others, which indicated that they were c-schizandrin, espatulenol, angeloylgomisin H, tigloylgo- the main targets of antioxidant functions. Among the 36 misin H, schisantherin B, gomisin K1, and gomisin M1, pathways, pathways in cancer, choline metabolism in cancer, which were colored yellow, were unique components in SCF and proteoglycans in cancer showed larger nodes than that were not observed in SSF. In addition, five compounds, others, which indicated that they were the main pathways namely, angeloylgomisin H, tigloylgomisin H, angeloylgo- through which the abovementioned bioactive compounds misin P, schisantherin C, and schisantherin, showed larger exerted antioxidant function. nodes than others, which indicated that they were the main As a dual-specific kinase, mitogen-activated protein antioxidant components. Among the 20 targets, four, kinase kinase 1 (MAP2K1) participates in the extracellular PC 2 (13.0%) Evidence-Based Complementary and Alternative Medicine 7 384.1931 Schisandrin 346.1411 369.1696 342.1459 415.2117 328.1303 400.1880 353.1744 333.1281 373.1649 359.1471 316.1299 309.1491 300 320 340 360 380 400 420 440 m/z (a) 384.1931 Sample 346.1410 369.1696 342.1453 328.1310 415.2113 400.1872 353.1750 333.1317 359.1479 373.1652 316.1310 300 320 340 360 380 400 420 440 m/z (b) Figure 3: -e MS spectrum of schisandrin in the standard and sample solution. regulated protein kinase (ERK) pathway by activating ERK1 hepatitis C. -e pathways related to MAPK8 include the and ERK2. -rough the identification of transforming genes ATM (serine/threonine kinase) pathway and the link be- for refractory diseases in the sclerosing subtypes of highly tween physicochemical characteristics and toxicity-related invasive tumors of gastric cancer, it was determined that pathways. Previous studies showed that MAPK8 is involved cancer cells are dependent on the increased proliferation in the gene expression process of anterior papillary hy- activity of MAP2K1, and MAP2K1 could be utilized as a pertension caused by hypoxia [31]. cancer inhibitor target [30]. PRKCB (protein kinase C) has been identified as a drug MAPK8 (mitogen-activated protein kinase 8), which is a treatment target for specific diseases involved in various protein-coding gene, was observed to participate in the cellular processes, such as the regulation of B-cell receptor abovementioned 16 pathways and to connect to 9 targets in (BCR) signaling bodies, oxidative stress-induced apoptosis, the PPI network diagram, which indicated its importance. male hormone receptor-dependent transcriptional regula- MAPK8-associated diseases include fatty liver disease and tion, insulin signaling, and endothelial cell proliferation. Relative abundance Relative abundance 8 Evidence-Based Complementary and Alternative Medicine +H OCH H CO H CO + H CO OCH H CO 3 CH H CO H CO CH + OCH H CO 3 3 H CO m/z 415.2113 CH H CO H CO 3 CH H CO H CO CH 3 3 m/z 384.1931 –H O 2 CH H CO m/z 400.1872 –OCH +H –CH –H O OCH H CO –H O H CO H CO CH H CO CH H CO 3 HO –H O –H O m/z 433.2213 –OCH –OCH –OCH –CH –H O OCH H CO OCH H CO –C H 4 8 CH H CO OCH H CO 3 CH H CO 3 CH H CO 3 3 H CO H CO CH 3 3 H CO m/z 369.1696 m/z 353.1750 H CO H CO m/z 359.1479 Figure 4: -e proposed fragmentation pathways of schisandrin. Table 3: Targets of potential antioxidant active compounds. No. UniProt ID Gene name Protein 1 P07339 CTSD Cathepsin D 2 Q16665 HIF1A Hypoxia-inducible factor 1 alpha 3 P06213 INSR Insulin receptor 4 P45983 MAPK8 c-Jun N-terminal kinase 1 5 P04629 NTRK1 Nerve growth factor receptor Trk-A 6 P05362 ICAM1 Intercellular adhesion molecule 1 7 P04035 HMGCR HMG-CoA reductase (by homology) 8 P05771 PRKCB Protein kinase C beta 9 Q02750 MAP2K1 Dual specificity mitogen-activated protein kinase kinase 1 10 P23219 PTGS1 Cyclooxygenase-1 11 Q01959 SLC6A3 Dopamine transporter (by homology) 12 P09917 ALOX5 Arachidonate 5-lipoxygenase 13 P16083 NQO2 Quinone reductase 2 14 P14416 DRD2 Dopamine D2 receptor (by homology) 15 P00813 ADA Adenosine deaminase 16 P23443 RPS6KB1 Ribosomal protein S6 kinase 1 17 P09237 MMP7 Matrix metalloproteinase 7 18 P05186 ALPL Alkaline phosphatase, tissue-nonspecific isozyme 19 P24385 CCND1 Cyclin-dependent kinase 4/cyclin D1 20 P49810 PSEN2 Gamma-secretase Evidence-Based Complementary and Alternative Medicine 9 MAPK8 MMP7 MAP2K1 ALPL NTRK1 INSR ADA PRKCB ICAM1 CTSD PTGS1 HIF1A PSEN2 HMGCR RPS6KB1 DRD2 NQO2 CCND1 SLC6A3 ALOX5 Figure 5: Protein-protein interaction network. Protein kinase activity Axon Membrane raft Response to nutrient levels Behavior Count Response to inorganic substance Response to toxic substance Response to wounding Aging Positive regulation of programmed cell death Positive regulation of ERK1 and ERK2 cascade Response to hypoxia Positive regulation of cell cycle Regulation of neuron apoptotic process NegLog10_Qvalue Regulation of response to wounding Response to glucocorticoid –0.2 Peptidyl-threonine phosphorylation –0.4 Collagen metabolic process Positive regulation of reactive oxygen species metabolic –0.6 process Response to nicotine 20 30 40 Sample group GO biological processes GO cellular components GO molecular functions Figure 6: Target biological enrichment analysis. PRKCB, as the locus of systemic lupus erythematosus [32], indicator of tumorigenesis and tumor progression. In- was observed to have upregulated mRNA expression in the creased levels of MAPK8 and HIF1A could induce the ex- peripheral blood mononuclear cells of patients with systemic pression of related genes and subsequently affect the cell lupus erythematosus. growth and proliferation [33]. -erefore, it could be deduced MAPK8 and HIF1A were important targets in choline that the differential compounds might help to treat cancer by metabolism. Abnormal choline metabolism may serve as an acting on MAPK8 and HIF1A. 10 Evidence-Based Complementary and Alternative Medicine Wnt signaling pathway yroid hormone signaling pathway yroid cancer TNF signaling pathway Sphingolipid signaling pathway Serotonergic synapse Ras signaling pathway Rap1 signaling pathway Proteoglycans in cancer Prolactin signaling pathway Pathways in cancer Pancreatic cancer Non-small cell lung cancer Neurotrophin signaling pathway Natural killer cell mediated cytotoxicity MAPK signaling pathway Insulin signaling pathway Insulin resistance Influenza A Inflammatory mediator regulation of TRP channels Hepatitis B HIF-1 signaling pathway GnRH signaling pathway Glioma Gap junction FoxO signaling pathway Focal adhesion Fc gamma R-mediated phagocytosis Fc epsilon RI signaling pathway ErbB signaling pathway Dopaminergic synapse Colorectal cancer Choline metabolism in cancer Central carbon metabolism in cancer Acute myeloid leukemia AMPK signaling pathway 02 46 Gene count Figure 7: KEGG enrichment pathway analysis. Figure 8: Compound-target-pathway network. As a cyclin, CCND1 participates in a variety of gene of lymphocyte malignant tumors, rack mounting, and expression and protein pathways. CCND1-associated multiple occurrences. CCND1 and its catalytic partner chromosomal aberrations could cause multiple occurrences cyclin-dependent kinase 4 (cdk4) play important roles in the Evidence-Based Complementary and Alternative Medicine 11 G1/S checkpoint of the cell cycle. Takano et al. [34] hy- UPLC/QEO MS: Ultrahigh-performance liquid pothesized that the synergistic effect of CCND1 and cdk4 chromatography-Q-Exactive Orbitrap with ER may cause breast cancer. mass spectrometry -rough pathway analysis, it was observed that the CTD: Comparative Toxicogenomics Database antioxidant activities of SSF and SCF were closely related to CTSD: Cathepsin D the HIF-1 (hypoxia-inducible factor 1) signaling pathway, HIF1A: Hypoxia-inducible factor 1-alpha pathways in cancer, and choline metabolism in cancer. HIF- INSR: Insulin receptor 1 is a transcription factor and a major regulator of oxygen MAPK8: Mitogen-activated protein kinase 8 homeostasis. -is protein consists of an inducible HIF-1- NTRK1: High-affinity nerve growth factor alpha subunit and a constitutively expressed HIF-1beta receptor subunit. Under normoxia and hypoxia, the conversion of the ICAM1: Intercellular adhesion molecule 1 two subunits regulates their transcriptional activity. Under HMGCR: 3-Hydroxy-3-methylglutaryl-coenzyme hypoxia, HIF-1 is the main regulator of many hypoxia-in- A reductase duced genes. HIF-1-related factors may cause such diseases PRKCB: Protein kinase C beta type as diabetic retinopathy, glucocorticoid-induced osteonec- MAP2K1: Mitogen-activated protein kinase kinase 1 rosis, and malignant paraganglioma [35]. PTGS1: Prostaglandin G/H synthase 1 Antioxidants were frequently used for cancer treatment SLC6A3: Sodium-dependent dopamine in previous studies [36], and angeloylgomisin H was re- transporter ported to be cytotoxic to human cancer cell lines [37], which ALOX5: Arachidonate 5-lipoxygenase suggested that antioxidant active compounds might also NQO2: Quinone reductase 2 possess anticancer activity. Among the 36 pathways obtained DRD2: Dopamine receptor D2 in this study, 9 related to pathways in cancer accounted for ADA: Adenosine deaminase 25%, which indicated thatSCF andSSF might serve as cancer RPS6KB1: Ribosomal protein S6 kinase beta-1 treatments by exerting antioxidant activity. MMP7: Matrix metalloproteinase 7 ALPL: Alkaline phosphatase, tissue- 5.Conclusion nonspecific isozyme CCND1: Cyclin-dependent kinase 4/cyclin D1 In this study, the antioxidant activity of SSF and SCF was PSEN2: Presenilin-2 assessed and compared by DPPH free radical scavenging RSD: Relative standard deviation percentage experiments, and the differential compounds, as well as their UV: Ultraviolet-visible spectrophotometer associated biological functions, were obtained by coupling UPLC-MS: Ultra high-performance liquid UPLC/Q-Exactive Orbitrap MS technology with network chromatography-mass spectrum in pharmacological analysis. -e results showed that the an- series tioxidant ability ofSCF was stronger than that ofSSF, and 14 PCA: Principal component analysis differential compounds were identified between SCF and NIST Chemistry National Institute of Standards and SSF, which indicated that these compounds were potential WebBook: Technology (NIST) Chemistry active components with antioxidant activity. Further anal- WebBook ysis showed that there were a total of 20 predicted targets and PPI: Protein-protein interaction 36 pathways related to these active components with anti- GO: Gene Ontology oxidant activity, and most of these targets and pathways KEGG: Kyoto Encyclopedia of Genes and were associated with cancer regulation. -e pharmacology Genomes network predicted that the differential components might DAVID: Database for Annotation, Visualization, exert antioxidant effects on the 20 targets by regulating the and Integrated Discovery 36 pathways. -ese medicines may be helpful for treating AMPK: AMP-activated protein kinase various diseases, especially cancer, by exerting antioxidant MAPK: Mitogen-activated protein kinase; activity. -erefore, this study provided a novel method for TNF: Tumor necrosis factor identifying active components with antioxidant activity, and FoxO: Forkhead box O this technique may be applicable for the filtration of bio- ERK: Extracellular regulated protein kinase active components in other herbs. ERK1: Extracellular regulated protein kinase 1 ERK2: Extracellular regulated protein kinase 2 Abbreviations TCMSP: Traditional Chinese Medicine Systems Pharmacology Database SSF: Schisandrae Sphenantherae Fructus ATM: Serine/threonine kinase SCF: Schisandrae Chinensis Fructus BCR: B-cell receptor DPPH: 1,1-Diphenyl-2-picrylhydrazyl CDK4: Cyclin-dependent kinase 4 VC: L-Ascorbic acid ER: Estrogen receptor IC : Half maximal inhibitory concentration 50 12 Evidence-Based Complementary and Alternative Medicine [9] Y.-Q. Zhang, X. Mao, Q.-Y. Guo, N. Lin, and S. Li, “Network HIF-1: Hypoxia-inducible factor 1 pharmacology-based approaches capture essence of Chinese GnRH: Gonadotropin-releasing hormone herbal medicines,” Chinese Herbal Medicines, vol. 8, no. 2, ErbB: Epidermal growth factor receptor. pp. 107–116, 2016. [10] G. B. Zhang, Q. Y. Li, Q. L. Chen, and S. B. Su, “Network Data Availability pharmacology: a new approach for Chinese herbal medicine research,” Evidence-based Complementary and Alternative -e data used to support the findings of this study are Medicine:ECAM, vol. 2013, Article ID 621423, 2013. available from the corresponding author upon request. [11] Y. Zhang, H. Y. Wang, Y. H Zhou, H. K Feng, and X. Q. Chen, “Study on the extraction of Schisandra chinensis Baill poly- saccharides by ultrasonic wave and hot water method,”Special Conflicts of Interest Wild Economic Animal and Plant Research, vol. 37, no. 3, pp. 52–57, 2015. -e authors declare that they have no conflicts of interest. [12] W. Li, Y. G. Song, K. Y Liu et al., “Rapid identification of the different constituents in Fructus Schisandrae Chinensis before Authors’ Contributions and after processing by UHPLC-QTOF/MS∼E combining with metabonomics,” Acta Pharmaceutica Sinica, vol. 51, Y. Xin and H. J. Wang conceived and designed the exper- no. 9, pp. 1445–1450, 2016. iments. Y. Yang and K. C. Yu performed the experiments. [13] S. Yang, L. Shan, H. Luo, X. Sheng, J. Du, and Y. Li, “Rapid Y. Xin and Y. Yang analyzed the data. Y. Xin and Y. Yang classification and identification of chemical components of wrote the manuscript. schisandra chinensis by UPLC-Q-TOF/MS combined with data post-processing,”Molecules, vol. 22, no.10, p.1778, 2017. Acknowledgments [14] Z. Lou, H. Zhang, C. Gong et al., “Analysis of lignans in Schisandra chinensis and rat plasma by high-performance -is study was supported by the National Natural Science liquid chromatography diode-array detection, time-of-flight Foundation of China (no. 81403067), the Fundamental mass spectrometry and quadrupole ion trap mass spec- Research Funds in Heilongjiang Provincial Universities (no. trometry,” Rapid Communications in Mass Spectrometry, vol. 23, no. 6, pp. 831–842, 2009. UNPYSCT-2017160), and the Postdoctoral Program in [15] H. Liu, H. Lai, X. Jia et al., “Comprehensive chemical analysis Heilongjiang Province (no. LBH-Z19099). of Schisandra chinensis by HPLC-DAD-MS combined with chemometrics,”Phytomedicine, vol. 20, no.12, pp.1135–1143, References [16] X. Huang, F. Song, Z. Liu, and S. Liu, “Studies on lignan [1] D. Harman, “Aging: a theory based on free radical and ra- constituents from Schisandra chinensis (Turcz.) Baill. fruits diation chemistry,” Journal of Gerontology, vol. 11, no. 3, using high-performance liquid chromatography/electrospray pp. 298–300, 1956. ionization multiple-stage tandem mass spectrometry,” Jour- [2] S. Kannan, “Free radical theory of autoimmunity,”Beoretical nal of Mass Spectrometry, vol. 42, no. 9, pp. 1148–1161, 2007. Biology and Medical Modelling, vol. 3, no. 1, p. 22, 2006. [17] S. Y. Lu, F. R. Song, and Z. Q. Lu, Traditional Chinese [3] W. A. Pryor, “Free radical biology: xenobiotics, cancer, and Medicine Mass Spectrometry Analysis, pp. 185–210, Science aging,” Annals of the New York Academy of Sciences, vol. 393, Press, Beijing, China, 2012. no. 1, pp. 1–22, 1982. [18] J. Zhang, J. Chen, Z. Liang, and C. Zhao, “New lignans and [4] S. Vijayalaxmi, S. K. Jayalakshmi, and K. Sreeramulu, their biological activities,” Chemistry & Biodiversity, vol. 11, “Polyphenols from different agricultural residues: extraction, no. 1, pp. 1–54, 2014. identification and their antioxidant properties,” Journal of [19] J. Liu, X. Zhang, F. Yang, T. Li, D. Wei, and Y. Ren, Food Science and Technology, vol. 52, no. 5, pp. 2761–2769, “Antimetastatic effect of a lipophilic ascorbic acid derivative with antioxidation through inhibition of tumor invasion,” [5] A. Szopa, R. Ekiert, and H. Ekiert, “Current knowledge of Cancer Chemotherapy and Pharmacology, vol. 57, no. 5, Schisandrachinensis (Turcz.) Baill. (Chinese magnolia vine) as pp. 584–590, 2006. a medicinal plant species: a review on the bioactive compo- [20] H.-M. Chao, M.-J. Chuang, J.-H. Liu et al., “Baicalein protects nents, pharmacological properties, analytical and biotech- against retinal ischemia by antioxidation, antiapoptosis, nological studies,” Phytochemistry Reviews, vol. 16, no. 2, downregulation of HIF-1α, VEGF, and MMP-9 and upre- pp. 195–218, 2017. gulation of HO-1,” Journal of Ocular Pharmacology and [6] Z. Li, X. He, F. Liu, J. Wang, and J. Feng, “A review of Berapeutics, vol. 29, no. 6, pp. 539–549, 2013. polysaccharides from Schisandra chinensis and Schisandra [21] J. Styskal, H. Van Remmen, A. Richardson, and A. B. Salmon, sphenanthera: properties, functions and applications,” Car- “Oxidative stress and diabetes: what can we learn about in- bohydrate Polymers, vol. 184, pp. 178–190, 2018. [7] X. B. Zuo, X. M. Sun, C. Y. Wang, J. Liu, X. H. Xiao, and sulin resistance from antioxidant mutant mouse models?” Free Radical Biology and Medicine, vol. 52, no. 1, pp. 46–58, Z. F. Bai, “Investigation on protective effect and efficacy difference of extract of Schisandrae Sphenantherae Fructus 2012. [22] E. J. Henriksen, “Exercise training and the antioxidant and Schisandrae Chinensis Fructus against acetaminophen- induced liver injury,” China Journal of Chinese Materia α-lipoic acid in the treatment of insulin resistance and type 2 diabetes,” Free Radical Biology and Medicine, vol. 40, no. 1, Medica, vol. 44, no. 6, pp. 1238–1245, 2019. [8] A. L. Hopkins, “Network pharmacology: the next paradigm in pp. 3–12, 2006. [23] E. S. Namiduru, M. Namiduru, M. Tarakçioglu, ˘ and A. Toy, drug discovery,” Nature Chemical Biology, vol. 4, no. 11, pp. 682–690, 2008. “Antioxidant defense in patients with chronic viral hepatitis B Evidence-Based Complementary and Alternative Medicine 13 and C type,”ClinicalLaboratory, vol. 56, no. 5-6, pp. 207–213, [24] L. Liang, X. L. Shou, H. K. Zhao et al., “Antioxidant catalase rescues against high fat diet-induced cardiac dysfunction via an IKKβ-AMPK-dependent regulation of autophagy,” Bio- chimicaetBiophysicaActa(BBA)—MolecularBasisofDisease, vol. 1852, no. 2, pp. 343–352, 2015. [25] S. U. Kim, Y. H. Park, J. S. Min et al., “Peroxiredoxin I is a ROS/p38 MAPK-dependent inducible antioxidant that reg- ulates NF-κB-mediated iNOS induction and microglial acti- vation,” Journal of Neuroimmunology, vol. 259, no. 1-2, pp. 26–36, 2013. [26] Z. B. Zamora, A. Borrego, O. Y. Lopez et al., “Effects of ozone oxidative preconditioning on TNF-α release and antioxidant- prooxidant intracellular balance in mice during endotoxic shock,” Mediators of Inflammation, vol. 2005, no. 1, 7 pages, Article ID 634736, 2005. [27] J. Kim, N. Ishihara, and T. R. Lee, “A DAF-16/FoxO3 a-dependent longevity signal is initiated by antioxidants,” BioFactors, vol. 40, no. 2, pp. 247–257, 2014. [28] Y. Lu and D.-F. Chen, “Analysis of Schisandra chinensis and Schisandra sphenanthera,” Journal of Chromatography A, vol. 1216, no. 11, pp. 1980–1990, 2009. [29] X. Chen, R. Tang, T. Liu et al., “Physicochemical properties, antioxidant activity and immunological effects in vitro of polysaccharides from Schisandra sphenanthera and Schisan- dra chinensis,” International Journal of Biological Macro- molecules, vol. 131, pp. 744–751, 2019. [30] Y. L. Choi, M. Soda, T. Ueno et al., “Oncogenic MAP2K1 mutations in human epithelial tumors,” Carcinogenesis, vol. 33, no. 5, pp. 956–961, 2012. [31] X. Zhang, W. Zhang, S.-F. Ma et al., “Hypoxic response contributes to altered gene expression and precapillary pul- monary hypertension in patients with sickle cell disease,” Circulation, vol. 129, no. 16, pp. 1650–1658, 2014. [32] Z. Zhu, L. Yang, Y. Zhang et al., “Increased expression of PRKCB mRNA in peripheral blood mononuclear cells from patients with systemic lupus erythematosus,” Annals of Hu- man Genetics, vol. 82, no. 4, pp. 200–205, 2018. [33] K. Glunde, Z. M. Bhujwalla, and S. M. Ronen, “Choline metabolism in malignant transformation,” Nature Reviews Cancer, vol. 11, no. 12, pp. 835–848, 2011. [34] Y. Takano, H. Takenaka, Y. Kato et al., “Cyclin D1 over- expression in invasive breast cancers: correlation with cyclin- dependent kinase 4 and oestrogen receptor overexpression, and lack of correlation with mitotic activity,” Journal of Cancer Research and Clinical Oncology, vol. 125, no. 8-9, pp. 505–512, 1999. [35] G. L. Semenza, “Oxygen homeostasis,”WileyInterdisciplinary Reviews: Systems Biology and Medicine, vol. 2, no. 3, pp. 336–361, 2010. [36] A. R. Collins, “Antioxidant intervention as a route to cancer prevention,” European Journal of Cancer, vol. 41, no. 13, pp. 1923–1930, 2005. [37] S. K. Choi, Y. G. Lee, R. B. Wang, H. G. Kim, and N. I. Baek, “Dibenzocyclooctadiene lignans from the fruits of Schisandra chinensis and their cytotoxicity on human cancer cell lines,” Applied Biological Chemistry, vol. 63, no. 1, 2020. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Evidence-Based Complementary and Alternative Medicine Hindawi Publishing Corporation

Filtration of Active Components with Antioxidant Activity Based on the Differing Antioxidant Abilities of Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus through UPLC/MS Coupling with Network Pharmacology

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Copyright © 2021 Yang Xin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 5547976, 13 pages https://doi.org/10.1155/2021/5547976 Research Article Filtration of Active Components with Antioxidant Activity Based on the Differing Antioxidant Abilities of Schisandrae Sphenantherae Fructus and Schisandrae Chinensis FructusthroughUPLC/MSCouplingwithNetworkPharmacology 1,2,3 1 1 4 Yang Xin , Yang Yang, Kaichen Yu, and Haijun Wang College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar 161006, China Heilongjiang Academy of Chinese Medical Sciences, Harbin 150036, China Heilongjiang Provincial Key Laboratory of Catalytic Synthesis for Fine Chemicals, Qiqihar University, Qiqihar 161006, China School of General Medicine and Continuing Education, Qiqihar Medical University, No. 333, Bukui Street, Qiqihar 161006, China Correspondence should be addressed to Yang Xin; cc.xinyang@163.com and Haijun Wang; whjxy0802@163.com Received 19 January 2021; Revised 20 June 2021; Accepted 7 July 2021; Published 22 July 2021 Academic Editor: Hicham Harhar Copyright © 2021 Yang Xin et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. -is study attempted to filter active components with antioxidant activities based on the differing antioxidant abilities of Schisandrae Sphenantherae Fructus (SSF) and Schisandrae Chinensis Fructus (SCF). First, the antioxidant activity of SSF and SCF was evaluated through the DPPH free radical scavenging method and compared with the half maximal inhibitory concentration (IC ) value. Next, components of SSF and SCF were detected by employing ultrahigh-performance liquid chromatography-Q- Exactive Orbitrap mass spectrometry (UPLC-QEO/MS) technology, and differential compounds were screened out as potential antioxidant compounds by using Compound Discover 3.1 Software. After that step, in order to verify the antioxidant compounds, the network method was applied. Biological targets were searched in the GeneCards database, and that related to antioxidant ability were selected in the Comparative Toxicogenomics Database (CTD). Finally, the pharmacology network was constructed. Results showed that SSF and SCF possessed different compounds and antioxidant abilities. A total of 14 differential compounds such as c-schizandrin, schisandrin B, schisandrin, and tigloylgomisin H between them were screened out and identified. Twenty targets associated with antioxidant activity contained MAP2K1, MAPK8, RPS6KB1, PRKCB, HIF1A, and so on were investigated. -irty-six pathways contained HIF-1 signaling pathways, choline metabolism in cancer, serotonergic synapse, Fc epsilon RI signaling pathway, GnRH signaling pathway, and so on related to the above twenty targets were identified. -e pharmacology network analysis indicated that the differential components may be helpful in treating various diseases, especially cancer, by exerting antioxidant activity. In conclusion, this study provided a novel method for identifying active components with anti- oxidant activity in SSF and SCF, and this method may be applicable for the filtration of bioactive components in other herbs. Free radicals are constantly produced in the body due to 1.Introduction exposure to the environment through processes such as Antioxidation is a process that can effectively inhibit the respiration (oxidation reaction), air pollution, and radiation. oxidation reaction caused by free radicals. -e mechanism of Free hydroxyl radicals are associated with aging stress [1], antioxidation involves either an action on free radicals or the autoimmunity [2], bone loss, and cancer development [3]. consumption of substances that can easily generate free However, substances that possess antioxidant activity can radicals. terminate or inhibit the oxidation process by scavenging free 2 Evidence-Based Complementary and Alternative Medicine radicals [4]. -us, maintaining the balance of free radicals (Alabama, USA), and L-ascorbic acid (vitamin C (VC)) was and the antioxidant active substance in the body can delay purchased from InnoCHEM (InnoCHEM, China). Ana- aging. -erefore, finding antioxidant active components is of lytical-grade anhydrous ethanol was used (Tianli, China). significance for developing antioxidant health products and pharmaceuticals to cure diseases caused by oxidative 2.2. Sample Preparation. SCF and SSF were prepared using damage, as well as for studying the mechanisms governing the reflux extraction process [11]; the extraction method for antioxidant activity. them was optimized based on the DPPH radical clearance Schisandrae Sphenantherae Fructus (SSF) and Schisan- rate by utilizing an L9(3 ) orthogonal design. During or- drae Chinensis Fructus (SCF) are both well-known Chinese thogonal experiments, factors concerning extract times, herbal medicines, and SSF is primarily produced in the reflux time, and material-liquid ratio were all inspected with central and southern part of China, whereasSCF is primarily four levels. -e optimal extraction process was as follows: produced in northern China. Although these medicines first, SCF or SSF was weighed 2.5g. Next, the herbal med- possess some similar pharmacological actions, such as an- icine was mixed with 20 volumes of water and then boiled for tioxidant, anti-inflammatory, and anxiolytic effects [5], SCF 2h, 5 times. -e extract was then filtered, mixed, and and SSF exhibit differences in chemical components [6] and concentrated to 50mL. After that, 2mL of the extract was other pharmacological actions [7], which might be the measured and dried. Finally, the residue was dissolved using reason why SCF is more popular in Chinese medicine than 2mL anhydrous ethanol before detection. SSF. -erefore, conducting a study simultaneously com- paring the components and biological activities, as well as their connections, was of interest. 2.3.DPPHRadicalClearanceTest. DPPH was dissolved with Regarding Chinese herbal medicine, because of the 95% ethanol aqueous solution to prepare 0.1mmol/L of multiple components of these medicines, the interactions DPPH solution and 2mL of it was mixed with 2mL sample. between them and treated organisms is very complex, which -e mixture was then detected at 517nm using a UV makes studies on the mechanisms governing the medicines spectrometer after 30min. difficult to conduct. Fortunately, the concept of “network pharmacology” was proposed by Hopkins in 2008 [8] and has been widely employed in research on Chinese herbal 2.4. Condition of Detection. -e absorbance of the SCF and medicine [9]. With the help of network pharmacology, the SSF extracts was measured on an ultraviolet-visible spec- correlation between drugs and action targets could be trophotometer TU1901 (Persee Co., China) in the spectral predicted through computer virtual computation, network scan mode in the range of 510∼516nm. database retrieval, network modeling, and network analysis. Compounds inSCF andSSF were detected on UPLC-MS Compared to traditional pharmacology, network pharma- equipment consisting of a Dionex UltiMate 3000 UHPLC cology exhibits the features of “multitarget, multieffect, and and Q-Exactive Orbitrap MS with an electrospray source in complex disease” [10]. Based on the above characteristics, positive-ion mode (-ermo Co., USA). network pharmacology has been recognized by an increasing UPLC separation was performed on a TM C18 column number of scholars, and network pharmacology has been (2.1 ×50mm, 2.6μm, -ermo Co., USA). Formic acid was increasingly utilized to study pharmacological effects. added to the mobile phase, which consisted of methanol (A) In this study, a network pharmacology method was and 0.1% formate water (B), to improve ionization efficacy employed to verify the antioxidant active compounds fil- and acquire a better peak shape. -e flow rate was set at −1 tered by comparing the components in SCF with those of 0.3mL·min . UPLC resolution was optimized with gradient SSF based on the results of UPLC/MS technology. Specifi- elution as follows: 0∼2min, 40%∼60% A; 2∼6min, 60%∼ cally, this report presents a novel method for determining 100% A; and 6∼8 min, 100% A. -e column temperature was the antioxidant components of these medicines, which 25 C, and the injection volume was 0.5µL. depends on connecting the differing antioxidant abilities of -e optimal mass spectrometry signal was obtained as SCF and SSF with the differential components between follows: capillary (+4.0kV), desolvation temperature them, as well as verifying these results through the strategy of (350 C), S-lens voltage (50V), shield gas (35arb), aux gas network pharmacology. -e method described in this study (10arb), scan type (full scan), and scan range (100∼1200Da). may facilitate the identification of antioxidant compounds in -e collision energy for MS was set at 10, 30, and 40eV to other herbal medicines. acquire abundant fragment ions. -e UV method was confirmed by inspecting the ac- curacy and stability of the absorbance of the same SCF 2.Experiment sample six times. -e stability was determined by testing six 2.1. Solvent and Medicine. Schisandrae Chinensis Fructus times in 30min. A relative standard deviation percentage (Schisandra chinensis (Turcz.) Baill., fruit, dried) was pur- (RSD%) of absorbance below 1% was considered to indicate chased from Qi Tai Pharmacy (Qiqihar, China). Schisandrae a good detection method. Sphenantherae Fructus (Schisandra sphenanthera Rehd. et -e UPLC-MS method was validated by inspecting the Wils., fruit, dried) was purchased from Xing Kang Pharmacy accuracy and stability through the detection of six com- (Qiqihar, China). -e reference standard for 1,1-diphenyl-2- pounds in both SCF and SSF. -e accuracy was determined picrylhydrazyl (DPPH) was purchased from Avanti by injecting the same SCF sample continuously six times. Evidence-Based Complementary and Alternative Medicine 3 -e stability was implemented by injecting the same SCF the accuracy and stability of the analytical method were sample at 0, 12, 24, 32, 40, and 48h. -e RSD% of retention acceptable. time and peak area below 5% was considered to indicate a good detection method. 3.2. Filtration of the Optimal Extraction Process for Antioxi- dantActivity. -e results of the orthogonal test are shown in Table 1. It can be seen that, through the optimization of 2.5.DataAnalysis. -e UV absorbance data of SSF and SCF the extract method, sample prepared by the extraction were used to calculate the DPPH radical clearance, as well as process with the material-liquid ratio of 1 : 20, extracted the IC value. First, the DPPH radical clearance was cal- for 5 times and 2.0 h/time showed the optimal DPPH culated as (Abs −Abs )/Abs ×100%. Second, the blank sample blank radical clearance rate, which is used for the extraction of IC value was acquired through curve fitting, with extract SSF and SCF. concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0mg/mL serving as the X-axis and the DPPH radical clearance value serving as the Y-axis, by GraphPad software. -ird, the IC values of 3.3.EstimationofAntioxidantActivitybetweenSSFandSCF. the medicines were compared with that of vitamin C to -e DPPH radical clearance ability of SSF and SCF was estimate their antioxidant ability. estimated by comparing the IC value with vitamin C (VC); All UPLC/MS raw data were acquired by Q-Exactive the lower the IC value was, the better the antioxidant Tune (-ermo Co., USA) and processed with Compound activity was. -e results showed that the IC values of SCF, Discoverer 3.1 (-ermo Co., USA). Next, a data list con- SSF, and VC were 0.4518±0.0008mg/mL, 0.4928± taining data such as molecular weight, retention time, peak 0.0015mg/mL, and 0.2225±0.0037mg/mL, respectively, intensity, and p which indicated thatSCF exhibited better antioxidant ability value of SCF versus SSF was acquired. Compounds with than SSF. a p value<0.05 were considered to be significantly different between SCF and SSF. Principal component analysis (PCA) 3.4.FilterationandIdentificationofthePotentialAntioxidant was used to observe the overall classification of the data. Active Components. To determine the reason for the anti- Next, the differential compounds were identified by oxidant ability of SSF being better than that of SCF, it was searching the mzCloud, ChemSpider, PubChem, NIST surmised that there were some compounds that showed a Chemistry WebBook databases, and references based on higher intensity inSCF than inSSF; therefore, the differential their MS spectra. compounds between SSF and SCF were filtered. -e targets of differential compounds were screened out First, samples of SSF and SCF were detected by UPLC/ in the Swiss Target Prediction database after the canonical MS, and total chromatograms were obtained using SMILES number was obtained from PubChem and com- Q-Exactive Tune (-ermo Co.). -e clear difference in the pounds were standardized by the UniProt database. -e peak amount and intensity between SSF and SCF is depicted CTD and GeneCards databases were searched for the targets in Figure 1. of antioxidant function. After that step, the common targets Second, the overall difference between SSF and SCF was of differential compounds and antioxidant function were observed through unsupervised principal component imported into the STRING database, and “multiple pro- analysis (PCA) (Figure 2), and variables making significant teins” and “human sources” were checked. Next, protein- contributions to discrimination between them were selected protein interactions (PPIs) were depicted using Cytoscape. based on a p value<0.05 by using Compound Discoverer 3.1 Next, Gene Ontology (GO) and Encyclopedia of Genes and software. Next, the Traditional Chinese Medicine Systems Genomes (KEGG) enrichment analyses were performed Pharmacology Database (TCMSP, https://tcmspw.com/ using the Metascape database. Finally, a network pharma- tcmsp.php), mzCloud (https://www.mzcloud.org/), Chem- cology diagram was drawn with the help of Cytoscape Spider (https://www.chemspider.com/), PubChem (https:// software. pubchem.ncbi.nlm.nih.gov/), and the National Institute of Standards and Technology (NIST) Chemistry WebBook (https://webbook.nist.gov/chemistry/) were searched to de- 3.Results termine the molecular weight and the molecular formula of different variables, and their MS 3.1. Evaluation of Methodology. Both of the methodologies spectra were compared with for UV and UPLC/MS were performed. For UV, the RSD% those in references to identify the chemical structures. A total of accuracy and stability was less than 0.5%. Regarding the of 14 compounds were identified, and all their intensity ratios UPLC/MS method, the RSD% accuracies for schisandrin, in SCF versus SSF were greater than 1; therefore, they were gomisin J, schisantherin B, angeloylgomisin P, schilancifo- initially considered antioxidant compounds, as shown in lignan A, and schisantherin C were 0.97%, 1.64%, 1.84%, Table 2. 1.72%, 0.85%, and 2.04%, respectively, and the RSD% of Among these compounds, espatulenol was proposed by stability for them in SSF was 2.55%, 2.44%, 2.90%, 0.68%, comparing the MS spectrum with that in the NIST 2.98%, and 3.52%, respectively, whereas those in SCF were Chemistry WebBook database, c-schizandrin, schisantherin 3.36%, 3.60%, 0.45%, 3.24%, 4.95%, and 0.91%, respectively. B, schisantherin C, tigloylgomisin H, gomisin J, ange- All the RSD% values were less than 5%, which indicated that loylgomisin P, angeloylgomisin H, gomisin M1, gomisin E, 4 Evidence-Based Complementary and Alternative Medicine Table 1: L9(3 ) orthogonal array design matrix and experimental 3.6. Protein-Protein Network Construction. -e protein- results. protein interaction (PPI) network diagram was constructed using Cytoscape software (Figure 5). Figure 5 shows that the No. A B (g/mL) C (/h) D DPPH clearance% network graph contained targets and 24 edges. Each target 1 3 (1) 1 :08 (1) 1.5 (1) 1 22.81 connected several functions. -e larger the degree value was, 2 3 (1) 1:10 (2) 2.0 (2) 2 27.34 the larger the node was, and the larger the combined score 3 3 (1) 1:20 (3) 2.5 (3) 3 27.50 value was, the thicker the edge was. Among these genes, the 4 4 (2) 1 :08 (1) 2.0 (2) 3 26.88 top 3 genes with larger nodes included MAPK8, CCND1, 5 4 (2) 1:10 (2) 2.5 (3) 1 26.72 6 4 (2) 1:20 (3) 1.5 (1) 2 30.63 and RPS6KB1, which indicated that they were important 7 5 (3) 1 :08 (1) 2.5 (3) 2 30.47 targets on which the potential antioxidant components 8 5 (3) 1:10 (2) 1.5 (1) 3 30.16 acted. 9 5 (3) 1:20 (3) 2.0 (2) 1 41.39 K1 25.883 26.720 27.867 30.307 — K2 28.077 28.073 31.870 29.480 — 3.7. Gene Ontology Enrichment Analysis for Targets. GO K3 34.007 33.173 28.230 28.180 — (gene ontology) enrichment analysis of potential targets was R 8.124 6.453 4.003 2.127 — performed using the Metascape database, and the functions Note: A: reflow times; B: material-liquid ratio; C: reflow time; D: blank. were enriched under the conditions of p value <0.01 and enrichment factor >1.5. Figure 6 shows the top 20 most significant functions. In Figure 7, the point and gomisin K1 were proposed by comparing the MS size represents the number of genes with the same function; spectrum with that in references [12–17], and schilancifo- the larger the point size is, the more genes there are. -e lignan A was speculated based on its MS fragments [18]. results showed that potential antioxidant compounds af- Schisandrin and schisandrin B were verified by their stan- fected numerous biological functions involving biological dards. -e ion of m/z 455.2040, which was one significantly processes, cellular components, and molecular functions, different compound betweenSSF andSCF, was selected as an such as the regulation of aging, the active regulation of example to illustrate the compound identification process. programmed cell death, the positive regulation of reactive First, the extract ion spectrum of m/z 455.2040 was acquired oxygen metabolism, the regulation of neuronal apoptosis, at t 3.71min. -en, its MS spectrum was acquired under positive regulation of the cell cycle, and regulation of the 40eV, which is shown in Figure 3. Figure 3 shows that collagen metabolism process. -ese targets might slow the fragment ions ofm/z 415.2113, 400.1872, 384.1931, 369.1696, occurrence and development of aging and hypoxia caused by 359.1479, and 353.1750 exerted the same fragmentation with the oxidation process by participating in the process of that of schisandrin reported in references, corresponded to + + + antioxidant reactions. [M+H-H O] , [M+H-H O-CH ] , [M+H-H O-OCH ] , 2 2 3 2 3 + + [M+H-H O-OCH -CH ] , [M+H-H O-C H ] , and 2 3 3 2 4 8 [M+H-H O-OCH -OCH ] based on high-resolution mass 2 3 3 3.8. Pathway Analysis for Targets. -e top 36 enriched spectrometry. Hence, it was deduced to schisandrin initially. pathways of the above 20 targets were obtained from the Further, the schisandrin standard was detected by the same DAVID (Database for Annotation, Visualization, and In- UPLC/MS condition for verifying the deduction, which tegrated Discovery) database, which is shown in Figure 7. showed that their retention time and MS spectrum were -e length of the column represents the number of genes matched completely. -erefore, the ion of 455.2040 was participating in the pathway; the longer the column is, the identified as schisandrin. -e proposed fragmentation more genes there are. Among these pathways, pathways in pathways of schisandrin are shown in Figure 4. cancer, the HIF-1 signaling pathway, insulin resistance, the insulin signaling pathway, hepatitis B, the AMPK signaling pathway, the MAPK signaling pathway, the TNF signaling 3.5. Prediction of the Targets of Antioxidant Compounds. pathway, and the FoxO signaling pathway were reported to A total of 441 targets on which the abovementioned potential be related to antioxidant activity [19–27]. antioxidant compounds acted were obtained. At the same time, 4151 targets associated with antioxidant activity were obtained by searching the GeneCards database, and 3946 of 3.9. Construction of Compound-Target-Pathway Network. them were retained after UniProt processing. Furthermore, -e compound-target-pathway network was constructed by 4531 targets related to antioxidant activity were obtained by connecting 14 differential compounds, 20 targets, and 36 searching the CTD database, and 150 of them were retained pathways. Figure 8 shows that there were a total of 70 nodes after UniProt processing. -ere were 147 common targets in and 230 edges. -e larger the node was, the more nodes were the GeneCards database and CTD database. Furthermore, connected to it. -erefore, angeloylgomisin H, aigloylgo- the 147 targets were compared to the abovementioned 441 misin H, angeloylgomisin P, schisantherin C, and schisan- targets, and it was found that 20 targets on which the an- therin B were deemed to have stronger antioxidant activity tioxidant compounds acted were related to antioxidant than other compounds. MAP2K1, MAPK8, PRKCB, and activity; these targets are listed in Table 3. CCND1 were considered to be the primary antioxidant effect Evidence-Based Complementary and Alternative Medicine 5 5.08 0.47 90 4.38 3.51 6.49 4.49 6.82 4.07 9.20 7.10 5.01 5.68 0.66 2.06 3.38 1.91 3.15 9.09 9.40 0.84 6.40 9.58 1.25 2.71 10.10 7.79 8.37 0 123456 7 8 9 10 11 Time (min) (a) 8.72 1.09 7.30 7.78 2.17 0.63 10 10.16 9.06 9.66 3.44 9.47 3.14 3.37 6.20 1.92 2.07 9.10 5.67 0.46 0.48 9.12 4.60 6.80 4.49 9.19 9.23 7.10 6.48 5.08 4.35 (b) Figure 1: Mirror image of the total ion chromatogram for SCF (a) and SSF (b). effects, the main effects that were essential for quality dif- targets. Pathways in cancer, choline metabolism in cancer, and proteoglycans were considered to be the primary ferences were not clear. -e antioxidant activity or com- pathways involved in antioxidant processes. ponents of medicines have long been compared by medicinal scholars [28, 29], but to the best of our knowledge, studies on the relationship of antioxidant ability and differential 4.Discussion components have not been performed to date. -erefore, this study described their relationship and predicted the SSF and SCF, which serve as typical “medicine-food ho- mology” herbal medicines, showed similar pharmacological functional targets of antioxidant activities using a network pharmacology strategy. -e compound-target-pathway effects, such as antioxidant activity. Nevertheless, SCF was generally more popular thanSSF because it was said thatSCF network diagram is shown in Figure 8, in which there were a total of 70 nodes and 230 edges. -e larger the degree value possessed higher quality than SSF. Regarding SSF and SCF, was, the larger the nodes were, and the stronger the because both of these medicines had multipharmacological 6 Evidence-Based Complementary and Alternative Medicine –10 –20 –60 –40 –20 0 20 40 PC 1 (44.5%) SSF SCF Figure 2: PCA for samples of SCF and SSF. Table 2: Components of potential antioxidant activities. Deduced t Elemental Ion Calculated Deviation No. MS compound (min) composition adduction mass (ppm) b,d + 1 c-Schizandrin 2.76 C H O [M+H] 401.1959 −0.5 386.1723, 370.1768, 359.1487, 355.1532, 337.1432 23 28 6 203.1795, 193.1587, 175.1481, 163.1481, 147.1168, b,d + 2 Espatulenol 3.40 C H O [M+H] 221.1900 −1.4 15 24 133.1012, 107.0858, 95.086 455.2032, 415.2113, 400.1872, 384.1931, 369.1696, a,b + 3 Schisandrin 3.71 C H O [M+Na] 455.2040 −2.0 24 32 7 359.1479, 353.1750 b,d + 4 Tigloylgomisin H 3.91 C H O [M+Na] 523.2302 −0.6 493.1786, 455.2077, 423.1418, 401.1621, 383.1506 28 36 8 [M+Na] 411.1778 −1.5 5 Gomisin J 3.99 C H O 357.1721, 325.1453, 319.1181, 297.1483, 287.0919 22 28 6 [M+H]+ 389.1943 −3.9 Schisantherin B or 6 4.09 C H O [M+Na] 537.2095 −0.7 437.1573,415.1750,371.1498,356.1246 b 28 34 9 angeloylgomisin P Angeloylgomisin P 7 4.12 C H O [M+H] 537.2095 −1.5 437.1573,415.1750,371.1498,356.1246 b 28 34 9 or schisantherin B Angeloylgomisin 416.1823, 387.1798, 372.1564, 356.1614, 342.1453, 8 4.13 C H O [M+Na] 523.2302 −0.6 b,d 28 36 8 H 326.1503 Schilancifolignan 9 4.25 C H O [M+H] 431.2064 −1.2 493.1840,455.2039,423.1422,383.1489 c,d 24 30 7 b + 10 Schisantherin C 4.50 C H O [M+H] 515.2275 −1.4 385.1649, 355.1543, 343.1183, 316.0944 28 34 9 b,d + 11 Gomisin K1 4.65 C H O [M+Na] 425.1934 −0.7 410.1701, 395.1464, 379.1516 23 30 6 b + 12 Gomisin E 4.97 C H O [M+H] 515.2275 −0.2 469.2206, 385.1646, 355.1538, 343.1179, 316.0941 28 34 9 b,d + 13 Gomisin M1 5.07 C H O [M+Na] 409.1621 −1.0 394.1393, 363.1185, 333.1724 22 26 6 a,b,d + 14 Schisandrin B 5.37 C H O [M+H] 401.1959 −0.7 300.0990,270.0883,242.0936,331.1174,386.1723,401.1953 23 28 6 -e superscript “a” represents compounds identified by standard, superscript “b” represents compounds identified by literature reports, superscript “c” represents compounds speculated based on its MS fragments, and superscript “d” represents compounds solely found in SCF. antioxidant activity of the compounds was. Among the 14 namely, MAP2K1, MAPK8, PRKCB, and CCND1, showed differential compounds, seven compounds, namely, larger nodes than the others, which indicated that they were c-schizandrin, espatulenol, angeloylgomisin H, tigloylgo- the main targets of antioxidant functions. Among the 36 misin H, schisantherin B, gomisin K1, and gomisin M1, pathways, pathways in cancer, choline metabolism in cancer, which were colored yellow, were unique components in SCF and proteoglycans in cancer showed larger nodes than that were not observed in SSF. In addition, five compounds, others, which indicated that they were the main pathways namely, angeloylgomisin H, tigloylgomisin H, angeloylgo- through which the abovementioned bioactive compounds misin P, schisantherin C, and schisantherin, showed larger exerted antioxidant function. nodes than others, which indicated that they were the main As a dual-specific kinase, mitogen-activated protein antioxidant components. Among the 20 targets, four, kinase kinase 1 (MAP2K1) participates in the extracellular PC 2 (13.0%) Evidence-Based Complementary and Alternative Medicine 7 384.1931 Schisandrin 346.1411 369.1696 342.1459 415.2117 328.1303 400.1880 353.1744 333.1281 373.1649 359.1471 316.1299 309.1491 300 320 340 360 380 400 420 440 m/z (a) 384.1931 Sample 346.1410 369.1696 342.1453 328.1310 415.2113 400.1872 353.1750 333.1317 359.1479 373.1652 316.1310 300 320 340 360 380 400 420 440 m/z (b) Figure 3: -e MS spectrum of schisandrin in the standard and sample solution. regulated protein kinase (ERK) pathway by activating ERK1 hepatitis C. -e pathways related to MAPK8 include the and ERK2. -rough the identification of transforming genes ATM (serine/threonine kinase) pathway and the link be- for refractory diseases in the sclerosing subtypes of highly tween physicochemical characteristics and toxicity-related invasive tumors of gastric cancer, it was determined that pathways. Previous studies showed that MAPK8 is involved cancer cells are dependent on the increased proliferation in the gene expression process of anterior papillary hy- activity of MAP2K1, and MAP2K1 could be utilized as a pertension caused by hypoxia [31]. cancer inhibitor target [30]. PRKCB (protein kinase C) has been identified as a drug MAPK8 (mitogen-activated protein kinase 8), which is a treatment target for specific diseases involved in various protein-coding gene, was observed to participate in the cellular processes, such as the regulation of B-cell receptor abovementioned 16 pathways and to connect to 9 targets in (BCR) signaling bodies, oxidative stress-induced apoptosis, the PPI network diagram, which indicated its importance. male hormone receptor-dependent transcriptional regula- MAPK8-associated diseases include fatty liver disease and tion, insulin signaling, and endothelial cell proliferation. Relative abundance Relative abundance 8 Evidence-Based Complementary and Alternative Medicine +H OCH H CO H CO + H CO OCH H CO 3 CH H CO H CO CH + OCH H CO 3 3 H CO m/z 415.2113 CH H CO H CO 3 CH H CO H CO CH 3 3 m/z 384.1931 –H O 2 CH H CO m/z 400.1872 –OCH +H –CH –H O OCH H CO –H O H CO H CO CH H CO CH H CO 3 HO –H O –H O m/z 433.2213 –OCH –OCH –OCH –CH –H O OCH H CO OCH H CO –C H 4 8 CH H CO OCH H CO 3 CH H CO 3 CH H CO 3 3 H CO H CO CH 3 3 H CO m/z 369.1696 m/z 353.1750 H CO H CO m/z 359.1479 Figure 4: -e proposed fragmentation pathways of schisandrin. Table 3: Targets of potential antioxidant active compounds. No. UniProt ID Gene name Protein 1 P07339 CTSD Cathepsin D 2 Q16665 HIF1A Hypoxia-inducible factor 1 alpha 3 P06213 INSR Insulin receptor 4 P45983 MAPK8 c-Jun N-terminal kinase 1 5 P04629 NTRK1 Nerve growth factor receptor Trk-A 6 P05362 ICAM1 Intercellular adhesion molecule 1 7 P04035 HMGCR HMG-CoA reductase (by homology) 8 P05771 PRKCB Protein kinase C beta 9 Q02750 MAP2K1 Dual specificity mitogen-activated protein kinase kinase 1 10 P23219 PTGS1 Cyclooxygenase-1 11 Q01959 SLC6A3 Dopamine transporter (by homology) 12 P09917 ALOX5 Arachidonate 5-lipoxygenase 13 P16083 NQO2 Quinone reductase 2 14 P14416 DRD2 Dopamine D2 receptor (by homology) 15 P00813 ADA Adenosine deaminase 16 P23443 RPS6KB1 Ribosomal protein S6 kinase 1 17 P09237 MMP7 Matrix metalloproteinase 7 18 P05186 ALPL Alkaline phosphatase, tissue-nonspecific isozyme 19 P24385 CCND1 Cyclin-dependent kinase 4/cyclin D1 20 P49810 PSEN2 Gamma-secretase Evidence-Based Complementary and Alternative Medicine 9 MAPK8 MMP7 MAP2K1 ALPL NTRK1 INSR ADA PRKCB ICAM1 CTSD PTGS1 HIF1A PSEN2 HMGCR RPS6KB1 DRD2 NQO2 CCND1 SLC6A3 ALOX5 Figure 5: Protein-protein interaction network. Protein kinase activity Axon Membrane raft Response to nutrient levels Behavior Count Response to inorganic substance Response to toxic substance Response to wounding Aging Positive regulation of programmed cell death Positive regulation of ERK1 and ERK2 cascade Response to hypoxia Positive regulation of cell cycle Regulation of neuron apoptotic process NegLog10_Qvalue Regulation of response to wounding Response to glucocorticoid –0.2 Peptidyl-threonine phosphorylation –0.4 Collagen metabolic process Positive regulation of reactive oxygen species metabolic –0.6 process Response to nicotine 20 30 40 Sample group GO biological processes GO cellular components GO molecular functions Figure 6: Target biological enrichment analysis. PRKCB, as the locus of systemic lupus erythematosus [32], indicator of tumorigenesis and tumor progression. In- was observed to have upregulated mRNA expression in the creased levels of MAPK8 and HIF1A could induce the ex- peripheral blood mononuclear cells of patients with systemic pression of related genes and subsequently affect the cell lupus erythematosus. growth and proliferation [33]. -erefore, it could be deduced MAPK8 and HIF1A were important targets in choline that the differential compounds might help to treat cancer by metabolism. Abnormal choline metabolism may serve as an acting on MAPK8 and HIF1A. 10 Evidence-Based Complementary and Alternative Medicine Wnt signaling pathway yroid hormone signaling pathway yroid cancer TNF signaling pathway Sphingolipid signaling pathway Serotonergic synapse Ras signaling pathway Rap1 signaling pathway Proteoglycans in cancer Prolactin signaling pathway Pathways in cancer Pancreatic cancer Non-small cell lung cancer Neurotrophin signaling pathway Natural killer cell mediated cytotoxicity MAPK signaling pathway Insulin signaling pathway Insulin resistance Influenza A Inflammatory mediator regulation of TRP channels Hepatitis B HIF-1 signaling pathway GnRH signaling pathway Glioma Gap junction FoxO signaling pathway Focal adhesion Fc gamma R-mediated phagocytosis Fc epsilon RI signaling pathway ErbB signaling pathway Dopaminergic synapse Colorectal cancer Choline metabolism in cancer Central carbon metabolism in cancer Acute myeloid leukemia AMPK signaling pathway 02 46 Gene count Figure 7: KEGG enrichment pathway analysis. Figure 8: Compound-target-pathway network. As a cyclin, CCND1 participates in a variety of gene of lymphocyte malignant tumors, rack mounting, and expression and protein pathways. CCND1-associated multiple occurrences. CCND1 and its catalytic partner chromosomal aberrations could cause multiple occurrences cyclin-dependent kinase 4 (cdk4) play important roles in the Evidence-Based Complementary and Alternative Medicine 11 G1/S checkpoint of the cell cycle. Takano et al. [34] hy- UPLC/QEO MS: Ultrahigh-performance liquid pothesized that the synergistic effect of CCND1 and cdk4 chromatography-Q-Exactive Orbitrap with ER may cause breast cancer. mass spectrometry -rough pathway analysis, it was observed that the CTD: Comparative Toxicogenomics Database antioxidant activities of SSF and SCF were closely related to CTSD: Cathepsin D the HIF-1 (hypoxia-inducible factor 1) signaling pathway, HIF1A: Hypoxia-inducible factor 1-alpha pathways in cancer, and choline metabolism in cancer. HIF- INSR: Insulin receptor 1 is a transcription factor and a major regulator of oxygen MAPK8: Mitogen-activated protein kinase 8 homeostasis. -is protein consists of an inducible HIF-1- NTRK1: High-affinity nerve growth factor alpha subunit and a constitutively expressed HIF-1beta receptor subunit. Under normoxia and hypoxia, the conversion of the ICAM1: Intercellular adhesion molecule 1 two subunits regulates their transcriptional activity. Under HMGCR: 3-Hydroxy-3-methylglutaryl-coenzyme hypoxia, HIF-1 is the main regulator of many hypoxia-in- A reductase duced genes. HIF-1-related factors may cause such diseases PRKCB: Protein kinase C beta type as diabetic retinopathy, glucocorticoid-induced osteonec- MAP2K1: Mitogen-activated protein kinase kinase 1 rosis, and malignant paraganglioma [35]. PTGS1: Prostaglandin G/H synthase 1 Antioxidants were frequently used for cancer treatment SLC6A3: Sodium-dependent dopamine in previous studies [36], and angeloylgomisin H was re- transporter ported to be cytotoxic to human cancer cell lines [37], which ALOX5: Arachidonate 5-lipoxygenase suggested that antioxidant active compounds might also NQO2: Quinone reductase 2 possess anticancer activity. Among the 36 pathways obtained DRD2: Dopamine receptor D2 in this study, 9 related to pathways in cancer accounted for ADA: Adenosine deaminase 25%, which indicated thatSCF andSSF might serve as cancer RPS6KB1: Ribosomal protein S6 kinase beta-1 treatments by exerting antioxidant activity. MMP7: Matrix metalloproteinase 7 ALPL: Alkaline phosphatase, tissue- 5.Conclusion nonspecific isozyme CCND1: Cyclin-dependent kinase 4/cyclin D1 In this study, the antioxidant activity of SSF and SCF was PSEN2: Presenilin-2 assessed and compared by DPPH free radical scavenging RSD: Relative standard deviation percentage experiments, and the differential compounds, as well as their UV: Ultraviolet-visible spectrophotometer associated biological functions, were obtained by coupling UPLC-MS: Ultra high-performance liquid UPLC/Q-Exactive Orbitrap MS technology with network chromatography-mass spectrum in pharmacological analysis. -e results showed that the an- series tioxidant ability ofSCF was stronger than that ofSSF, and 14 PCA: Principal component analysis differential compounds were identified between SCF and NIST Chemistry National Institute of Standards and SSF, which indicated that these compounds were potential WebBook: Technology (NIST) Chemistry active components with antioxidant activity. Further anal- WebBook ysis showed that there were a total of 20 predicted targets and PPI: Protein-protein interaction 36 pathways related to these active components with anti- GO: Gene Ontology oxidant activity, and most of these targets and pathways KEGG: Kyoto Encyclopedia of Genes and were associated with cancer regulation. -e pharmacology Genomes network predicted that the differential components might DAVID: Database for Annotation, Visualization, exert antioxidant effects on the 20 targets by regulating the and Integrated Discovery 36 pathways. -ese medicines may be helpful for treating AMPK: AMP-activated protein kinase various diseases, especially cancer, by exerting antioxidant MAPK: Mitogen-activated protein kinase; activity. -erefore, this study provided a novel method for TNF: Tumor necrosis factor identifying active components with antioxidant activity, and FoxO: Forkhead box O this technique may be applicable for the filtration of bio- ERK: Extracellular regulated protein kinase active components in other herbs. ERK1: Extracellular regulated protein kinase 1 ERK2: Extracellular regulated protein kinase 2 Abbreviations TCMSP: Traditional Chinese Medicine Systems Pharmacology Database SSF: Schisandrae Sphenantherae Fructus ATM: Serine/threonine kinase SCF: Schisandrae Chinensis Fructus BCR: B-cell receptor DPPH: 1,1-Diphenyl-2-picrylhydrazyl CDK4: Cyclin-dependent kinase 4 VC: L-Ascorbic acid ER: Estrogen receptor IC : Half maximal inhibitory concentration 50 12 Evidence-Based Complementary and Alternative Medicine [9] Y.-Q. Zhang, X. Mao, Q.-Y. Guo, N. Lin, and S. Li, “Network HIF-1: Hypoxia-inducible factor 1 pharmacology-based approaches capture essence of Chinese GnRH: Gonadotropin-releasing hormone herbal medicines,” Chinese Herbal Medicines, vol. 8, no. 2, ErbB: Epidermal growth factor receptor. pp. 107–116, 2016. [10] G. B. Zhang, Q. Y. Li, Q. L. Chen, and S. B. Su, “Network Data Availability pharmacology: a new approach for Chinese herbal medicine research,” Evidence-based Complementary and Alternative -e data used to support the findings of this study are Medicine:ECAM, vol. 2013, Article ID 621423, 2013. available from the corresponding author upon request. [11] Y. Zhang, H. Y. Wang, Y. H Zhou, H. K Feng, and X. Q. Chen, “Study on the extraction of Schisandra chinensis Baill poly- saccharides by ultrasonic wave and hot water method,”Special Conflicts of Interest Wild Economic Animal and Plant Research, vol. 37, no. 3, pp. 52–57, 2015. -e authors declare that they have no conflicts of interest. [12] W. Li, Y. G. Song, K. Y Liu et al., “Rapid identification of the different constituents in Fructus Schisandrae Chinensis before Authors’ Contributions and after processing by UHPLC-QTOF/MS∼E combining with metabonomics,” Acta Pharmaceutica Sinica, vol. 51, Y. Xin and H. J. Wang conceived and designed the exper- no. 9, pp. 1445–1450, 2016. iments. Y. Yang and K. C. Yu performed the experiments. [13] S. Yang, L. Shan, H. Luo, X. Sheng, J. Du, and Y. Li, “Rapid Y. Xin and Y. Yang analyzed the data. Y. Xin and Y. Yang classification and identification of chemical components of wrote the manuscript. schisandra chinensis by UPLC-Q-TOF/MS combined with data post-processing,”Molecules, vol. 22, no.10, p.1778, 2017. Acknowledgments [14] Z. Lou, H. Zhang, C. Gong et al., “Analysis of lignans in Schisandra chinensis and rat plasma by high-performance -is study was supported by the National Natural Science liquid chromatography diode-array detection, time-of-flight Foundation of China (no. 81403067), the Fundamental mass spectrometry and quadrupole ion trap mass spec- Research Funds in Heilongjiang Provincial Universities (no. trometry,” Rapid Communications in Mass Spectrometry, vol. 23, no. 6, pp. 831–842, 2009. UNPYSCT-2017160), and the Postdoctoral Program in [15] H. Liu, H. Lai, X. Jia et al., “Comprehensive chemical analysis Heilongjiang Province (no. LBH-Z19099). of Schisandra chinensis by HPLC-DAD-MS combined with chemometrics,”Phytomedicine, vol. 20, no.12, pp.1135–1143, References [16] X. Huang, F. Song, Z. Liu, and S. Liu, “Studies on lignan [1] D. Harman, “Aging: a theory based on free radical and ra- constituents from Schisandra chinensis (Turcz.) Baill. fruits diation chemistry,” Journal of Gerontology, vol. 11, no. 3, using high-performance liquid chromatography/electrospray pp. 298–300, 1956. ionization multiple-stage tandem mass spectrometry,” Jour- [2] S. Kannan, “Free radical theory of autoimmunity,”Beoretical nal of Mass Spectrometry, vol. 42, no. 9, pp. 1148–1161, 2007. Biology and Medical Modelling, vol. 3, no. 1, p. 22, 2006. [17] S. Y. Lu, F. R. Song, and Z. Q. Lu, Traditional Chinese [3] W. A. Pryor, “Free radical biology: xenobiotics, cancer, and Medicine Mass Spectrometry Analysis, pp. 185–210, Science aging,” Annals of the New York Academy of Sciences, vol. 393, Press, Beijing, China, 2012. no. 1, pp. 1–22, 1982. [18] J. Zhang, J. Chen, Z. Liang, and C. Zhao, “New lignans and [4] S. Vijayalaxmi, S. K. Jayalakshmi, and K. Sreeramulu, their biological activities,” Chemistry & Biodiversity, vol. 11, “Polyphenols from different agricultural residues: extraction, no. 1, pp. 1–54, 2014. identification and their antioxidant properties,” Journal of [19] J. Liu, X. Zhang, F. Yang, T. Li, D. Wei, and Y. Ren, Food Science and Technology, vol. 52, no. 5, pp. 2761–2769, “Antimetastatic effect of a lipophilic ascorbic acid derivative with antioxidation through inhibition of tumor invasion,” [5] A. Szopa, R. Ekiert, and H. Ekiert, “Current knowledge of Cancer Chemotherapy and Pharmacology, vol. 57, no. 5, Schisandrachinensis (Turcz.) Baill. (Chinese magnolia vine) as pp. 584–590, 2006. a medicinal plant species: a review on the bioactive compo- [20] H.-M. Chao, M.-J. Chuang, J.-H. Liu et al., “Baicalein protects nents, pharmacological properties, analytical and biotech- against retinal ischemia by antioxidation, antiapoptosis, nological studies,” Phytochemistry Reviews, vol. 16, no. 2, downregulation of HIF-1α, VEGF, and MMP-9 and upre- pp. 195–218, 2017. gulation of HO-1,” Journal of Ocular Pharmacology and [6] Z. Li, X. He, F. Liu, J. Wang, and J. Feng, “A review of Berapeutics, vol. 29, no. 6, pp. 539–549, 2013. polysaccharides from Schisandra chinensis and Schisandra [21] J. Styskal, H. Van Remmen, A. Richardson, and A. B. Salmon, sphenanthera: properties, functions and applications,” Car- “Oxidative stress and diabetes: what can we learn about in- bohydrate Polymers, vol. 184, pp. 178–190, 2018. [7] X. B. Zuo, X. M. Sun, C. Y. Wang, J. Liu, X. H. Xiao, and sulin resistance from antioxidant mutant mouse models?” Free Radical Biology and Medicine, vol. 52, no. 1, pp. 46–58, Z. F. Bai, “Investigation on protective effect and efficacy difference of extract of Schisandrae Sphenantherae Fructus 2012. [22] E. J. Henriksen, “Exercise training and the antioxidant and Schisandrae Chinensis Fructus against acetaminophen- induced liver injury,” China Journal of Chinese Materia α-lipoic acid in the treatment of insulin resistance and type 2 diabetes,” Free Radical Biology and Medicine, vol. 40, no. 1, Medica, vol. 44, no. 6, pp. 1238–1245, 2019. [8] A. L. Hopkins, “Network pharmacology: the next paradigm in pp. 3–12, 2006. [23] E. S. Namiduru, M. Namiduru, M. Tarakçioglu, ˘ and A. Toy, drug discovery,” Nature Chemical Biology, vol. 4, no. 11, pp. 682–690, 2008. “Antioxidant defense in patients with chronic viral hepatitis B Evidence-Based Complementary and Alternative Medicine 13 and C type,”ClinicalLaboratory, vol. 56, no. 5-6, pp. 207–213, [24] L. Liang, X. L. Shou, H. K. Zhao et al., “Antioxidant catalase rescues against high fat diet-induced cardiac dysfunction via an IKKβ-AMPK-dependent regulation of autophagy,” Bio- chimicaetBiophysicaActa(BBA)—MolecularBasisofDisease, vol. 1852, no. 2, pp. 343–352, 2015. [25] S. U. Kim, Y. H. Park, J. S. Min et al., “Peroxiredoxin I is a ROS/p38 MAPK-dependent inducible antioxidant that reg- ulates NF-κB-mediated iNOS induction and microglial acti- vation,” Journal of Neuroimmunology, vol. 259, no. 1-2, pp. 26–36, 2013. [26] Z. B. Zamora, A. Borrego, O. Y. Lopez et al., “Effects of ozone oxidative preconditioning on TNF-α release and antioxidant- prooxidant intracellular balance in mice during endotoxic shock,” Mediators of Inflammation, vol. 2005, no. 1, 7 pages, Article ID 634736, 2005. [27] J. Kim, N. Ishihara, and T. R. Lee, “A DAF-16/FoxO3 a-dependent longevity signal is initiated by antioxidants,” BioFactors, vol. 40, no. 2, pp. 247–257, 2014. [28] Y. Lu and D.-F. Chen, “Analysis of Schisandra chinensis and Schisandra sphenanthera,” Journal of Chromatography A, vol. 1216, no. 11, pp. 1980–1990, 2009. [29] X. Chen, R. Tang, T. Liu et al., “Physicochemical properties, antioxidant activity and immunological effects in vitro of polysaccharides from Schisandra sphenanthera and Schisan- dra chinensis,” International Journal of Biological Macro- molecules, vol. 131, pp. 744–751, 2019. [30] Y. L. Choi, M. Soda, T. Ueno et al., “Oncogenic MAP2K1 mutations in human epithelial tumors,” Carcinogenesis, vol. 33, no. 5, pp. 956–961, 2012. [31] X. Zhang, W. Zhang, S.-F. Ma et al., “Hypoxic response contributes to altered gene expression and precapillary pul- monary hypertension in patients with sickle cell disease,” Circulation, vol. 129, no. 16, pp. 1650–1658, 2014. [32] Z. Zhu, L. Yang, Y. Zhang et al., “Increased expression of PRKCB mRNA in peripheral blood mononuclear cells from patients with systemic lupus erythematosus,” Annals of Hu- man Genetics, vol. 82, no. 4, pp. 200–205, 2018. [33] K. Glunde, Z. M. Bhujwalla, and S. M. Ronen, “Choline metabolism in malignant transformation,” Nature Reviews Cancer, vol. 11, no. 12, pp. 835–848, 2011. [34] Y. Takano, H. Takenaka, Y. Kato et al., “Cyclin D1 over- expression in invasive breast cancers: correlation with cyclin- dependent kinase 4 and oestrogen receptor overexpression, and lack of correlation with mitotic activity,” Journal of Cancer Research and Clinical Oncology, vol. 125, no. 8-9, pp. 505–512, 1999. [35] G. L. Semenza, “Oxygen homeostasis,”WileyInterdisciplinary Reviews: Systems Biology and Medicine, vol. 2, no. 3, pp. 336–361, 2010. [36] A. R. Collins, “Antioxidant intervention as a route to cancer prevention,” European Journal of Cancer, vol. 41, no. 13, pp. 1923–1930, 2005. [37] S. K. Choi, Y. G. Lee, R. B. Wang, H. G. Kim, and N. I. Baek, “Dibenzocyclooctadiene lignans from the fruits of Schisandra chinensis and their cytotoxicity on human cancer cell lines,” Applied Biological Chemistry, vol. 63, no. 1, 2020.

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Evidence-Based Complementary and Alternative MedicineHindawi Publishing Corporation

Published: Jul 22, 2021

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