Systematically Characterizing Chemical Profile and Potential Mechanisms of Qingre Lidan Decoction Acting on Cholelithiasis by Integrating UHPLC-QTOF-MS and Network Target Analysis

Qingre Lidan Decoction (QRLDD), a classic precompounded prescription, is widely used as an effective treatment for cholelithiasis clinically. However, its chemical profile and mechanism have not been characterized and elucidated. In the present study, a rapid, sensitive, and reliable ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method was established for comprehensively identifying the major constituents in QRLDD. Furthermore, a network pharmacology strategy based on the chemical profile was applied to clarify the synergetic mechanism. A total of 72 compounds containing flavonoids, terpenes, phenolic acid, anthraquinones, phenethylalchohol glycosides, and other miscellaneous compounds were identified, respectively. 410 disease genes, 432 compound targets, and 71 related pathways based on cholelithiasis-related and compound-related targets databases as well as related pathways predicted by the Kyoto Encyclopedia of Genes and Genomes database were achieved. Among these pathways and genes, pathway in cancer and MAPK signaling pathway may play an important role in the development of cholelithiasis. EGFR may be a crucial target in the conversion of gallstones to gallbladder carcinoma. Regulation of PRKCB/RAF1/MAP2K1/MAPK1 is associated with cell proliferation and differentiation. Thus, the fingerprint coupled with network pharmacology analysis could contribute to simplifying the complex system and providing directions for further research of QRLDD.


Introduction
Traditional Chinese Medicine possess a history of thousands of years, which has been widely used in clinical practice in China and played an increasingly important role to health maintenance and disease treatment. Traditional Chinese Formula (TCF) is the main form of clinical application of Traditional Chinese Medicine. Due to its satisfactory clinical efficacy, TCF has been regarded as an alternative and promising medicine strategy for treating complex diseases all over the world [1]. Qingre Lidan Decoction (QRLDD) is a classic precompounded prescription, which contains 6 herbs, namely, Lysimachiae Herba (jin-qian-cao in Chinese), Scutellariae Radix (huang-qin in Chinese), Aurantii Fructus (zhi-qiao in Chinese), Aucklandiae Radix (mu-xiang in Chinese), Gardeniae Fructus (zhi-zi in Chinese), and Rhei Radix et Rhizoma (da-huang in Chinese). It has been extensively applied in clinical treatment of cholecystitis and gallstones for many years with the satisfactory therapeutic effects in several hospitals [2,3]. The main mechanism of its efficacy has been reported to relax sphincter of Oddi, promote bile excretion, and prevent stagnation [4]. However, the current research on QRLDD has two drawbacks: firstly, a clear understanding of the relationship between ingredient and formula has not been elucidated; secondly, in aspect of pharmaceutical effect, current reports usually focus on the level of single inflammatory mediator or protein, which is hardly to reflect the characteristic of multicomponents and multitargets of Chinese medicine formula [5]. These are obstacles for the development and the therapeutic efficacy of QRLDD. In recent years, the rapid development of network pharmacology has provided a novel method for revealing the molecular mechanisms associated with the therapeutic efficacy of multicomponent in TCF [6]. It has facilitated understanding the interactions of ingredient, target, and disease systematically based on systems biology, polypharmacology, and molecular network analysis, rather than an individual target [7]. Thus, the application of network pharmacology provides a powerful and promising method for analyzing TCF.
The schematic diagram of present study was shown in Figure 1; an ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) method was established to analyze the major chemical constituents of QRLDD in this present study. Potential targets and related pathways were correspondingly explored by using network pharmacology method based on the identified components, and the mechanism of QRLDD in the treatment of cholelithiasis was elucidated systematically.

Preparation of Samples and Standard Solution.
The QRLDD samples were prepared by the decocting method. A blended mixture of Lysimachiae Herba (30 g), Scutellariae Radix (15 g), Aucklandiae Radix (15 g), Aurantii Fructus (15 g), and Gardeniae Fructus (15 g) was soaked in 10-fold mass of water (900 mL) for 1 h and boiled for 1 h and then filtered with six-layer absorbent gauze. An 8-fold mass of water (800 mL) was subsequently added to residues and boiled for 30 min. Then Rhei Radix et Rhizoma (10 g) was added into the extract and boiled for additional 30 min. After being filtered with sixlayer absorbent gauze, the two filtrates were combined and concentrated under vacuum to 100 mL (equal to 1 g crude herb/mL), and finally the concentrate was transformed into the freeze-dried powder. A 1.0 g of the freeze-dried powder was accurately weighted and extracted with 50 mL of methanol/water (1:1, v/v) for 30 min under ultrasound. The extract solution was centrifuged at 13000 rpm for 10 min at 4 ∘ C, and the supernatant was filtered through a 0.22 m filter. 1.0 L of filtrate was injected to UHPLC-QTOF-MS for analysis.
Mass detection was performed using an Agilent 6530b Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer (Agilent Corp., USA) equipped with a Dual AJS ESI source operating in both positive and negative mode with the following operating parameters: drying gas (N 2 ) flow rate, 10.0 L/min; drying gas (N 2 ) temperature, 350 ∘ C; nebulizer, 35 psig; sheath gas (N 2 ) temperature, 400 ∘ C; fragmentor voltage, 120 V; skimmer voltage, 65 V; Octopole RF, 750 V. The capillary voltage was set at 4 kV or -3.5 kV under positive or negative mode, respectively. The nozzle voltage was set at +500 V or -1000 V, respectively; four collision energies at 10 V, 20 V, 30 V, and 40 V were applied to acquire sufficient product ions. MS spectra were recorded over the m/z range of 50-1100. All data was processed by MassHunter workstation software version B.06.00 (Agilent Technologies, Germany).

Compound Target for QRLDD.
After identifying the compounds contained in QRLDD by UHPLC-QTOF-MS/MS, the InChI Key, Canonical SMILES, and CAS number of compounds were obtained from NCBI PubChem database (https://www.ncbi.nlm.nih.gov/pubmed/). And ingredient-related targets were accordingly collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (http://lsp.nwu .edu.cn/tcmsp.php) and Swiss Target Prediction (http:// www.swisstargetprediction.ch/) with their names and/or CAS number as key words. Then, their official symbol was obtained after input of the targets with the species limited to "Homo sapiens" via UniProtKB (http://www.uniprot.org/) [14]. Finally, genes information of ingredients was achieved. The details are supplied in Supplementary Table S2.

The Protein-Protein Interactions (PPIs) Network
Analysis. The protein-protein interactions (PPIs) network was constructed and analyzed by STRING database. In order to further identify the primary therapeutic targets to guarantee the accuracy of results, only those PPIs with high confidence score (>0.95) were selected for network construction and analysis [15].

Network Construction and Analysis.
All the networks can be performed by utilizing the network visualization software Cytoscape 3.2.1 [16], which supplies a method for data integration, analysis, and visualization for complicated network analysis. Three networks were constructed as follows: (1) protein-protein interactions (PPIs) of cholelithiasis targets; (2) herb-compound-compound targets network of QRLDD; (3) pathways-targets network analysis. In this network plot, a "node" signifies an herb, ingredient, or gene; an "edge" represents interaction among different targets. The "degree" of a node was in agreement with the number of its connected edges [17].

Enrichment Analysis.
To clarify the pathways that are relate to putative QRLDD targets, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway aenrichments bsed on Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/home.jsp, ver. 6.8) were applied [18].

Chemical Profile of QRLDD by UHPLC-QTOF-MS.
In the present study, a specific UHPLC-ESI-QTOF MS n protocol was performed to rapidly identify the compounds of QRLDD based on the optimized LC and MS conditions systemically.
As a result, a total of 72 compounds, including 33 flavonoids, 17 terpene, 9 phenolic acid, 5 anthraquinones, 3 phenethylalchohol glycosides, and 5 miscellaneous compounds were identified or tentatively characterized ( Figure 2, Table 1). Among them, 23 constituents (compounds -, , , , , -, , , -, , , , -, , , and -) were unambiguously identified as gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, (+)-catechin, Evidence-Based Complementary and Alternative Medicine 7      Table 1   (1) Dihydroflavones. A total of seven dihydroflavones were identified from QRLD samples, with peaks , , and definitely elucidated and the others tentatively assigned. Peaks and were accurately identified as hesperidin and neohesperidin by compared with their respective references. Corresponding to the previous paper [19], highaccurate quasi-molecular ions of peak were obtained in negative ion mode at m/z 609.1823, which was identified as hesperidin. O) lower than that of Peak . Therefore, it was identified as narirutin, a methoxy-substituted derivative at C-6 position, according to the above information and literature [20]. Correspondingly, peaks , , , and were tentatively assigned as carthamidin, neoeriocitrin, naringin, and hesperetin based on in-house library for QRLDD and further fragmentation patterns mentioned above.  In addition, cyclization reaction was also observed in part of flavones and their glycosides.
Peak was selected as the example for the stepwise elucidation of this appearance. It was identified as rutin by comparing with authentic standard, which exhibited quasimolecular ion [M-H] − at m/z 609.1466. Its MS 2 spectra gave the ions at m/z 463.0896 and m/z 301.0346, indicating the successive loss of rhamnose and rutinose, while, except for similar skeleton with baicalein (Peak ), m/z 178 and m/z 151 generated by cyclization reaction after RDA reaction in the C ring were also observed in the MS/MS spectrum (Figure 3(c)). Analogically, the other compounds were tentatively assigned following this fragmentation pathway and related literatures.

Identification of Terpenes.
Seventeen terpenoids, including nine iridoids and their glycosides, three sesquiterpenoids, three diterpenes, and two monoterpenes, were screened from QRLDD. Among them, peaks and were unambiguously identified as geniposide and dehydrocostuslactone by comparison with reference standards.  (Figure 3(d)). Similarly, peak with a [M+HCOO] − ion at m/z 449.1300 (C 17 H 24 O 11 , retention time 3.58 min) was 16 Da (+O) higher than quasi-molecular ion of peak . It also produced a desugarization ion at m/z 225.0772. Its predominant fragment ions at m/z 139 and m/z 101 were obtained owing to the RDA reaction. The former was 16 Da (+O) higher than that of Peak . Thus, this compound was tentatively assigned as scandoside methyl ester according to publications [21]. Analogously, the remaining compounds were tentatively identified by comparison of their retention behavior and MS/MS spectrum with the literature date [21,22].
(2) Sesquiterpenoids. Two distinct peaks and with [M+H] + ions at m/z 233.1535 and 231.1357 were observed in positive ion mode, respectively. Their most probable molecular formulas were inferred to be C 15 H 20 O 2 and C 15 H 18 O 2 according to exact molecular weight. Compound was identified as dehydrocostuslactone by comparison with its standard. Its tandem mass spectra and possible fragmentation pathway was illustrated in Figure 3(e). It showed the protonated ion at m/z 231.1357. The fragment ions at m/z 213, 185, 157, 195, and 175 were the characteristic behavior owing to successive neutral losses of H 2 O, CH 2 O 2 , C 3 H 6 O 2 , H 2 O 4 , and C 4 H 8, respectively [23]. Compound was accordingly identified as costus lactone in a similar way. In addition, Peak from Scutellariae radix was observed in negative ion mode and identified as dikamaliartanes A on the basis of MS data and related literature [22]. , which was 162 Da (+C 6 H 10 O 5 ) higher than that of peak . They also showed the same fragments ions with Peak . By matching the constructed compound library, they were deduced to crocin-1 and crocin-4, a pair of cis-trans isomer originated from Gardeniae Fructus. In addition, as the polarity of cis-diterpenes was larger than that of trans-diterpenes, peaks and were identified as crocin-1 and crocin-4, respectively [22,24].
Two monoterpenoids from Gardeniae Fructus were tentatively identified and their cleavage pathway is similar to that of iridoid glycosides with slightly differences. The losses of glycosides (162 Da), CO 2 (44 Da), and H 2 O (18 Da) were the characteristic fragmentations in their MS 2 spectra [22,25]. Peak was selected as the example for the stepwise elucidation of the molecular structure. It yielded the ions at m/z 183.0996 and m/z 165.0923, which corresponded to successive losses of a glycoside and H 2 O, respectively. The former further produced a fragment ion at m/z 121.0626 [M-H-glc-CO 2 ] − . Consequently, Peak was reasonably deduced to be jasminoside B according to aforementioned fragmental information and reference data (Figure 3(f)) [22]. Peak was tentatively assigned as jasminoside S/H/I following this fragmentation pathway; however, it needed to be confirmed by the reference standards.

Identification of Phenolic Acids.
Nine phenolic acids, originated from Scutellariae Radix, Lysimachiae Herba, and Gardeniae Fructus, were detected as minority of components in QRLDD. The negative ion mode was much more suitable for their analysis. Peaks , , , , and were unambiguously identified as gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, chlorogenic acid, and caffeic acid by comparison with authentic references. Peaks and were tentatively identified as darendoside A and p-coumaric acid on the basis of the exact molecular formulae matching, fragmentation, and the literature date [22,26]. Take  were the same as chlorogenic acid ( ), suggesting that they should be isomers of chlorogenic acid. Tao et al. reported that three isomeric neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid were contained in Gardeniae Fructus [27]. Moreover, the retention time for chlorogenic acid was later and earlier than that of neochlorogenic acid and cryptochlorogenic acid in a similar UHPLC system, respectively [28]. Therefore, peaks and were tentatively identified as neochlorogenic acid and cryptochlorogenic acid, respectively.

Identification of Anthraquinones.
Five anthraquinones were unambiguously identified by comparison with authentic references, which were more suitable for the analysis in negative ion mode. Successive or simultaneous neutral losses of H 2 O, CO, O, and CH 3 were the characteristic behavior of this type of compounds. Peak (t R = 30.24 min) was selected as an example, which displayed the [M-H] − ion at m/z 269.0453. The yield ion at m/z 241.0511 was formed by direct loss of the CO, followed by the loss of O, and gave the ion at m/z 225. 0559. The fragment ion of m/z 181, 251, and 223 was corresponded to the losses of CO 2 , H 2 O, and CO, respectively (Figure 3(h)). Similarly, aloeemodin, rhein, chrysophanol, and physcion were elucidated [24].

Identification of Phenethylalchohol Glycosides.
Three phenethylalchohol glycosides were tentatively identified due to the absence of reference standards, which were from Scutellariae Radix and Lysimachiae Herba. Caffeic acid, hydroxytyrosol, and glycosyls were the basic groups of this type of compounds. Peak was selected as the example for the stepwise elucidation.  (Figure 3(i)). It was identified as acteoside in consistent with the fragment information of literature [29]. Analogously, the remaining peaks and were tentatively identified as isomers cistanoside D and cistanoside C following above fragmentation pathway and polarity feature [29].   Figure 5: Herb-compound-compound target network of QRLDD (blue circle represents compound targets, cyan diamond represents for compound, and green hexagon represents herb; node size represents the degree).

Other Types of Miscellaneous Compounds.
Other compounds (peaks , , , , and ) were tentatively assigned as galloyl glucose, procyanidin B 2 , syringin, meranzin, and limonin, respectively, on the basis of the exact molecular formulae matching, fragmentation information as well as the literature data [30][31][32] but still need to be further confirmed by reference standard.

Cholelithiasis-Related Targets Network
Analysis. The relationship among 410 disease genes from PPI was extracted by STRING. And a gene-gene interaction network was accordingly constructed. 122 nodes and 173 edges were involved in this network ( Figure 4). Among them, the nodes located at the central part (IL6, NFKB1 and STAT3) connected by more edges have higher degree, such as 13 in IL6, 13 in NFKB1, and 10 in STAT3. It implies that these genes may be the important targets in the formation and development of cholelithiasis.

Herb-Compound-Compound Targets Network Analysis.
The relationship among 432 compound targets from PPI were constructed and analyzed by STRING. Compound targets of PPI with high confidence score (>0.95) were screened. And herb-compound-compound targets network constructed by cytoscape was shown in Figure 5, which comprises 313 nodes ( Figure 7: Illustration of relations among chemical constituent targets and involved pathways of QRLDD (green circle represents compound target, blue triangle represents pathway, green hexagon represents herb, and purple hexagon represents pathway. Node size represents the degree).
Scutellariae Radix may be the main herbs in treating disease due to their higher degree. According to the frequency statistics of 77 Chinese medicine cases on gallstones, Lysimachiae Herba, Scutellariae Radix, Aurantii Fructus, and Aucklandiae Radix were used for 55, 47, 21, and 12 times, respectively [33]. We can also find that many compounds acting on the same target and multiple targets contacted by the same compound. For example, MAPT is the targets of aloe-emodin, geniposide, gallic acid, and other chemical components. Quercetin simultaneously acts on IL10, MAPK1, HSF1, among many other targets. However, some can be regulated by only one compound, such as CA9, which is simply controlled by Khelloside. The degree of top 20 targets was listed in Figure 6.
The result indicted that compounds from QRLDD may act on these targets systematically and play an important pharmacological role in treating cholelithiasis, which is in line with herbal formulae's feature of multicompound and multi-target. The potential mechanism can be elucidated by this network.

Pathway of QRLDD-Disease Network.
In order to better understand the mechanism of QRLDD on cholelithiasis, 71 related pathways (P<0.5) were obtained by inputting all targets into DAVID; the details are described in Supplementary Table S3. As shown in Figure 7, Pathway in cancer (hsa05200) is ranked first, which has 72 genes involved; among them, PTGS2, TP53, and IL6 have a higher degree. The result is consistent with clinical data, which confirmed the close relationship between gallstones and gallbladder cancer [34]. Within the screened genes, EGFR was selected for example, whose expression is associated with proliferation, differentiation, lymphatic metastasis, and other processes of gallbladder carcinoma [35]. Therefore, the analysis of pathways in cancer will help to understand the pathogenesis of gallbladder carcinoma caused by gallstones and provide basis for the future study. In addition, there were 41, 35, and 31 pathways associated with MAPK1, MAP2K1, and RAF1, among which, MAPK signaling pathway (hsa04010), chemokine signaling pathway (hsa04062), and Focal adhesion (hsa04510) were the most closely related ones. KEGG data visualization made it obvious that the PRKCB downstream target proteins RAF1, MAP2K1 and MAPK1 are key connection points between the MAPK signaling pathway, chemokine signaling pathway and focal adhesion. According to previous study, lithogenic diet is closely related to the development of cholelithiasis, which tends to alter the components in bile with increasing substances such as arachidonyl lecithin and dehydrocholesterol. As an adaptive response to the environment, cell turnover will be emerged [36]. In the development of gallstone formation, mitotic index is shown to increase rapidly at prelithiasic phase [37]. In addition, the condition of gallbladder and bile duct abnormalities has been shown to accelerate the cell turnover and increase cellular proliferating activity [38]. In our present research, regulation of PRKCB/RAF1/MAP2K1/MAPK1 can affect cell proliferation and differentiation (Figure 8). These changes may provide a new perspective for the treatment of cholelithiasis.
Above findings provide a direct connection between metabolic syndrome and cholesterol gallstone. Whether their expression is involved in the curative effect of QRLDD acting on cholelithiasis will be validated in the subsequent research.

Conclusions
Chinese medicine plays an important role in preventing and treating cholelithiasis. In our study, the chemical profile of Qingre Lidan Decoction was mapped for the first time by UHPLC-QTOF-MS, and 72 ingredients origin from six herbs were attributed. The "multicomponent-multitargetmultipath" mechanism of QRLDD was further explored based on network pharmacology platform in view of the identified ingredient. Our study found that multiple ingredients in QRLDD can exert a combined effect for the same target. Several important targets (EGFR and MAPK1) and pathways (pathways in cancer and MAPK signaling pathway) were predicted to be an important role in the mechanism of QRLDD. The present study not only provide experimental and theoretical basis for the further development and application of QRLDD, but also make beneficial exploration in investigating the molecular synergy of Traditional Chinese Formula.

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

Conflicts of Interest
All authors have no financial or scientific conflicts of interest in regard to the research described in this manuscript.