Illumination on Chemical Compounds from Qufeng Zhitong Capsule and Its Potential Pharmacological Mechanism against Rheumatoid Arthritis Based on UHPLC/Q-Orbitrap-MS Combined with Network Pharmacology Analysis

Qufeng Zhitong capsule (QZC), a Chinese patent medicine officially approved in China for the treatment of rheumatoid arthritis (RA) and other diseases, possesses the primary effects of dispelling wind, relieving pain, and promoting blood circulation, whose clinical applications have been confined owing to the incomplete elucidation of its chemical compositions and the underlying molecular mechanism for the treatment of RA. In this study, 61 compounds including 16 phenylpropanoids, 15 organic acids, 13 alkaloids, seven flavonoids, six iridoids, one saccharide, two aldehydes, and one saponin in QZC were simultaneously identified and traced to their herbal origins by ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap high-resolution mass spectrometry (UHPLC/Q-Orbitrap-MS), where 31 of them were unambiguously identified by reference compounds, and the other 30 were tentatively characterized. Besides, all these compounds were proven to have potential pharmacological activity in the treatment of RA based on network pharmacology analysis. In conclusion, this study first investigated the chemical composition and potential pharmacological effects of the main chemical compounds in QZC, which will contribute to the revelation of bioactive compounds in QZC and provide evidence for clinical application.


Introduction
Rheumatoid arthritis (RA) is a chronic infammatory joint disease of an autoimmune nature, featuring persistent infammatory synovitis, whose progression is infuenced by multiple factors including environmental, genetic, and infection factors [1][2][3]. With the accelerated pace of life and increased work pressure, the number of people sufering from RA in China is increasing annually, which is difcult to heal and prone to recurrence, seriously afecting their physical and mental health, and quality of life. Besides, nonsteroidal antiinfammatory drugs and slow-acting antirheumatic drugs, mainly adopted by western medicine, usually lead to gastrointestinal side efects and psychological disorders. RA belongs to the category of "obstinate rheumatism" in traditional Chinese medicine (TCM), which is usually treated by removing wind and cold, dispelling dampness and clearing heat, eliminating blood stasis, and promoting blood circulation with remarkable efcacy [4], where Qufeng Zhitong capsule (QZC) is one of the efective drugs listed in the national essential medicine for the treatment of RA [5].
QZC, an efective patent medicine for treating RA, is composed of seven herbs that play diferent roles. Herba Geranii (Laoguancao, HG) and Radix Aconiti Kusnezofi Cocta (Zhicaowu, RAKC) are monarch herbs that mainly exert the efect of dispelling wind and dampness, relaxing tendons, and relieving pain. Flos Carthami (Honghua, FC) and Radix Angelicae Pubescentis (Duhuo, RAP) serve as minister drugs with the function of dispelling wind and dredging collaterals, activating blood circulation and stimulating meridians, and reducing swelling and relieving pain. Radix Dipsaci (Xuduan, RD), Radix et Rhizoma Clematidis (Weilingxian, ReRC), and Herba Visci (Hujisheng, HV) all act as assistant drugs to possess the efects of dispelling wind and dampness, dredging collaterals and relieving pain, invigorating the liver and kidney, and strengthening tendons and bones [6]. With the integrated efects of herbs, QZC can be widely used to alleviate the symptoms of RA, such as limb numbness, waist and knee pain, owing to the synergism of these herbal medicines [7].
At present, most studies of QZC focus on its clinical applications with few analytical studies on its chemical compounds, causing poorly clarifed chemical substances. In addition, little attention has been paid to the identifcation and quality evaluation of the overall medical product. QZC, as a TCM prescription, also has the characteristics of multicomponents, multitargets, and multipathways, and its comprehensive action mechanism on RA is still unclear. Li et al. reported that QZC has a positive efect on cortical bone and can prevent bone erosion in patients with RA [8]. With the rapid development of bioinformatics, network pharmacology, characterized by the holistic idea of TCM, has become a powerful tool and has been successfully applied to forecast the complicated action mechanism of TCM in the remedy of multiple illnesses by constructing and analyzing biological networks. Using existing databases, the potential mechanisms of Chinese herbs can be predicted, which can provide a theoretical reference for in-depth studies on the underlying mechanisms of TCM prescriptions [9].
In this study, a fast and efective method was developed for the chemical characterization of QZC using ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap high-resolution mass spectrometry (UHPLC/Q-Orbitrap-MS). Te systematic pharmacological mechanism of QZC in the treatment of RA was predicted via network pharmacology. Tis is the frst study focusing on the analysis of the chemical types, herb origins, and potential pharmacological efects of the main chemical components in QZC, whose results can provide a reference for future research on the chemical compositions, pharmacological efects, and clinical applications of QZC.  , protocatechuic acid,   neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid,  loganic acid, corilagin, loganin, isochlorogenic acids A−C,  angelol A, columbianetin acetate, osthole, columbianadin,  akebia saponin D, 5-hydroxymethyl furfural, 4-hydroxybenzoic acid, cafeic acid, hydroxysafor yellow A, syringin, ferulic acid, secoxyloganin, ellagic acid, columbianetin, angelol G, and isoimperatorin. Benzoylmesaconine, benzoylaconine, and benzoylhypaconine were obtained from Shanghai Acmec Biochemical Co., Ltd. (Shanghai, China). Kaempferol-3-Orutinoside was obtained from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). Te purities of these reference compounds were all determined to be above 98% by UPLC analysis.

Sample Solution Preparation.
Te accurately weighed QZC powder (0.5 g) was transferred into a 25 mL volumetric fask and ultrasonically extracted with a certain amount of 75% methanol aqueous solution at 60°C for 30 min, which was subsequently diluted to scale by 75% methanol aqueous solution after cooling down to room temperature.

Characterization of Chemical Constituents in Qufeng Zhitong Capsule by UHPLC/Q-Orbitrap-MS.
Te chemical compounds of QZC were identifed by UHPLC/Q-Orbitrap-MS analysis in both positive and negative ion modes. As a result, 61 compounds were characterized by comparison with reported literatures, including 16 phenylpropanoids, 15 organic acids, 13 alkaloids, seven favonoids, six iridoids, one saccharide, two aldehydes, and one saponin, among which 31 compounds were confrmed by comparison with the reference substances. Te fragment ions of identifed compounds are shown in Table 1, and the MS spectra of QZC sample and reference compounds are displayed in Figure 1, respectively. Te MS spectra of individual herbs are shown in Figure 2. Te chemical structures of the compounds identifed by comparison with the reference substances in QZC are shown in Figure S1. Coumarins, which are widely distributed in the plant families of Rutaceae and Umbelliferae, have been reported to have various therapeutic properties, such as antibacterial, antitumor, and antidepression activities [22]. Coumarins in QZC were found to mainly come from Duhuo.  International Journal of Analytical Chemistry identifed as umbelliferone, nodakenetin, columbianetin, angelol D, angelol A, angelol B, angelol K, angelol G, columbianetin acetate, isoangenomalin, angenomalin, osthole, isoimperatorin, and columbianadin, respectively. With the continuous development of natural product chemistry and the constant improvement of analytical techniques, organic acid components in TCM, such as chlorogenic acid and gallic acid, have been discovered to have strong activity, drawing increasing attention globally. Organic acids refer to acidic compounds containing the carboxylic group (−COOH), mostly distributed in herbs with a sour taste, and have been demonstrated to possess various pharmacological activities, including anti-infammatory, antioxidation, and inhibition of platelet aggregation, which have important clinical application value in the prevention and treatment of cardiovascular diseases [23]. In negative ion mode, organic acids exhibited similar cleavage behaviours by the loss of CO 2 , H 2 O, or CO. Compounds 1,2,5,7,10,15,16,19,25,28,33,35,36,42, and 57 were identifed as quinic acid, gallic acid, protocatechuic acid, neochlorogenic acid, 4hydroxybenzoic acid, cafeic acid, chlorogenic acid, cryptochlorogenic acid, corilagin, ferulic acid, ellagic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and 9,12,13-trihydroxy-10-octadecenoic acid, respectively. Quinic acid is mainly present in Hujisheng.  [19].
Iridoids, a type of monoterpene derivative in TCM, are widely distributed and have a variety of biological activities, such as antitumor, antiviral, and anti-infammatory. Iridoids feature complex structures, mostly containing hemiacetal hydroxyl groups and cyclopentane rings, which are prone to the formation of iridoid glycosides with saccharides. Te loss of monosaccharides or monosaccharide residues and neutral molecules such as CO 2 (44), H 2 O (18), C 2 H 2 (26), and CH 2 O (30), usually occurs in the mass spectra of iridoids [18]. Te iridoids in QZC are mostly derived from XuDuan. In negative ion mode, compound 30  According to the quasimolecular ions and the secondary fragment ions information, the fragmentation patterns of iridoid glycosides were analyzed. Compounds 20,27,29,46, and 52 were identifed as loganic acid, sweroside, loganin, cantleyoside, and triplostoside A.
As an important ingredient in QZC, RAKC was reported to have alkaloids, such as aconine, mesaconine, and hypaconine, which were responsible for anti-infammation and analgesia functions. Notably, toxicity would happen when RAKC was misused, which must be concerned about. Te pharmacopoeia of the People's Republic of China stipulates that the total amount of aconitine, mesaconitine, and hypaconine must be no more than 0.04%, and the total amount of benzoylaconitine, benzoylhypaconine, and benzoylmesaconine must range from 0.02% to 0.07% [7]. Importantly, the dosage and the course of treatment of QZC must be paid more attention to in clinical use by taking the detected risky alkaloids from RAKC into account.
HG, RAP, RD, and FC are efective ingredients in QZC. From HG, corilagin was reported to exert antirheumatoid arthritis via downregulation of NF-κB and MAPK signaling pathways [26]. Te osthole in RAP can reduce proinfammatory cytokines, such as TNF-α, IL-1β, and IL-6, showing promising analgesic efects [27]. Hydroxysafor yellow A, which derived from FC, can reduce expression of infammatory mediators in synovial tissue, thus reducing infammation and synovitis [28]. In addition, asperosaponin VI, one of the saponins in RD, can signifcantly inhibit RANKL-induced osteoclast formation to relieve arthritis in vivo [29]. Te abovementioned compounds play an important role in the treatment of RA.

Network Pharmacology.
As an open-source database, the SwissTargetPrediction is a web-based tool which can   predict the targets of any bioactive small molecule as ligands [30]. IPA is a graphical interface bioinformatics software based on cloud computing, which can search for some important information, such as genes, proteins, diseases, and classical pathways [31]. Based on the 61 compounds identifed by UHPLC/Q-Orbitrap-MS, a network pharmacological analysis was carried out to preliminarily predict their potential pharmacological activities. Te chemical structures and InChIs for 61 compounds from QZC were obtained from the PubChem database and literatures, which were uploaded to the SwissTargetPrediction database to obtain the targets of HOMO except for hypaconine, angelol A, angelol B, angelol D, angelol G, angelol K, columbianetin, mesaconine, and nodakenetin. Te BATMAN-TCM database was used to predict the targets of the aforementioned nine compounds, with a cutof score of 3.351. A total of 1002 targets were acquired after removing duplicates, which were imported to IPA software for core analysis with 926 targets detected. Te 1002 targets corresponding to the 61 index compounds in QZC are shown in Table S2. RA was selected for analysis in the related diseases, resulting in 173 related targets, which are shown in Table S3. Te network of "QZC-componentstargets-RA" is shown in Figure 3(a). "Pathogen-infuenced signaling" was selected for the reason that pathogen infection is an important problem in the research of RA [32]. Te canonical pathway analysis for pathogen-infuenced signaling of the predicted QZC targets is shown in Figure 3(b).
Te pathogenesis of rheumatoid arthritis is still unclear. Current studies suggest that infammation caused by an imbalance of T1/T2 cytokines is an important pathological mechanism [33]. T1 cells mainly secrete proinfammatory cytokines, such as interferon-c (IFN-c) and interleukin-1 (IL-1), to promote the production of infammatory cytokines. T2 cells mainly secrete anti-infammatory cytokines such as interleukin-4 (IL-4) and transforming growth factor-β (TGFβ), and inhibit the production of infammatory cytokines [34]. T1 and T2 cells are balanced in healthy people, while T1 cells increase and T2 cells decrease in patients with RA [35]. NF-κB, ICAM-1, JAK, IL-6, IL-23, CCR1, and CCR3 are closely associated with the T1/T2 signaling pathway, which may be regulated by the chemical compounds from QZC, suggesting that the chemical compounds from QZC may participate in the balance of the T1/T2 signaling pathway, thus, playing a therapeutic role in RA.
Te results indicated that the identifed compounds detected by UHPLC/Q-Orbitrap-MS have potential pharmacological activity for the treatment of RA, which sheds light on the modern approaches to investigate QZC and promoting the development of QZC.

Conclusions
In this study, 61 compounds in QZC were identifed by UHPLC/Q-Orbitrap-MS, including 16 phenylpropanoids, 15 organic acids, 13 alkaloids, seven favonoids, six iridoids, one saccharide, two aldehydes, and one saponin, with their herbal sources traced. Among them, 31 compounds were unambiguously identifed by reference compounds, and another 30 compounds were tentatively characterized based on their fragmentation pathways. Network pharmacology analysis revealed that all detected compounds exhibited potential pharmacological activity in the treatment of RA via diferent signaling pathways. In conclusion, this is the frst study focusing on the analysis of the overall chemical basis of QZC, which is conducive to revealing the bioactive compounds and potential application value of QZC.

Data Availability
Te data used to support the fndings of this study are included within the article and the supplementary information fle.

Conflicts of Interest
Te authors declare that they have no conficts of interest.