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Dissection of the Active Ingredients and Potential Mechanism of Han-Shi-Yu-Fei-Decoction in Treating COVID-19 Based on In Vivo Substances Profiling and Clinical Symptom-Guided Network Pharmacology

This work was aimed to elucidate the mechanism of action of Han-Shi-Yu-Fei-decoction (HSYFD) for treating patients with mild coronavirus disease 2019 (COVID-19) based on clinical symptom-guided network pharmacology. Experimentally, an ultra-high performance liquid chromatography technique coupled with quadrupole time-of-flight mass spectrometry method was used to profile the chemical components and the absorbed prototype constituents in rat serum after its oral administration, and 11 out of 108 compounds were identified. Calculatingly, the disease targets of Han-Shi-Yu-Fei symptoms of COVID-19 were constructed through the TCMIP V2.0 database. The subsequent network pharmacology and molecular docking analysis explored the molecular mechanism of the absorbed prototype constituents in the treatment of COVID-19. A total of 42 HSYFD targets oriented by COVID-19 clinical symptom were obtained, with EGFR, TP53, TNF, JAK2, NR3C1, TH, COMT, and DRD2 as the core targets. Enriched pathway analysis yielded multiple COVID-19-related signaling pathways, such as the PI3K/AKT signaling pathway and JAK-STAT pathway. Molecular docking showed that the key compounds, such as 6-gingerol, 10-gingerol, and scopoletin, had high binding activity to the core targets like COMT, JAK2, and NR3C1. Our work also verified the feasibility of clinical symptom-guided network pharmacology analysis of chemical compounds, and provided a possible agreement between the points of views of traditional Chinese medicine and western medicine on the disease.


INTRODUCTION
Since the outbreak of COVID-19 in 2019, it has stayed a global public health concern that has not yet been effectively resolved, posing a serious threat to human health and economic development. 1 Traditional Chinese medicine (TCM) has played a vital role in COVID-19 prevention and treatment. It has been listed in the "Diagnosis and Treatment Plan for COVID-19 Infection", version 3, issued by the National Health Commission of the People's Republic of China. 2 Significantly, the combination of TCM and western medicine has led to significant achievements in the battle against COVID-19. 3 It is well accepted that COVID-19 may evolve in four (overlapping) phases, from mild to moderate to severe to critical, which is staged by the clinical symptoms. Treatment of patients in mild and moderate stages is of utmost importance because about 14% of patients can progress to severe stage in only 1 week. 4 Of note, both TCM and western medicine systems have a general agreement on this point. 5 The typical clinical symptoms, including fever, cough, abnormality of the pharynx, fatigue, anergy, dyspnea, abnormality of the stomach, nausea, vomiting, diarrhea, abnormality of the tongue, and abnormality of the vasculature, were interpreted as "Han" and "Shi" that were blocked in the lung, according to the theory of TCM. Therefore, the typical recommended TCM recipe was  6 However, its active compounds and therapeutic mechanisms have not been studied yet.
According to the current network of pharmacology research and a few in vitro experiments, the mechanism of TCM in COVID-19 is a multi-component, multi-target, and multipathway, which exhibits the functions of combating viral infections, immune regulation, amelioration of lung injury and fibrosis, and protection of target organs. 7,8 However, most of the network pharmacology research studies were guided by the disease targets. 9 As the COVID-19 patients were staged by the clinical symptoms, which were associated with the progression from mild to moderate COVID-19 illness to severe or critical status, it is an effective way to force mechanism study by the disease symptom-guided network pharmacology.
In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC−Q-TOF-MS) method was used to identify the chemical components of HSYFD in vitro and then discriminate the blood-absorbing constituents, which were involved in the   subsequent network pharmacology analysis and molecular docking techniques ( Figure 1). 10,11 This strategy, based on a UHPLC−Q-TOF-MS method combined with network pharmacology, can decrease false-positive results for the TCM which should be orally administered and could be widely applied to discover phytochemical or biomolecular evidence with distinct potential functional basis. Our work provides meaningful data for further pharmacological study of HSYFD in the treatment of COVID-19.

Optimization of UHPLC−Q-TOF-MS Conditions.
The separation of the chemical constituents was performed on a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) by gradient elution with a mobile phase (phase A: 0.1% aqueous formic acid solution and phase B: acetonitrile solution). 12,13 The chromatographic peaks were well separated and distributed in the total ion chromatogram (TIC) when the elution time was set at 30 min.

Identification of Compounds in HSYFD.
The TICs of HSYFD in both positive and negative modes are shown in Figure 2. This was the first step to construct an in-house compound database 1.0 (HSYFD) by combining the compound information. The available information including the chemical name, formula, and accurate molecular weight, by searching the reference databases (such as PubMed, SciFinder, Web of Science, and CNKI), and generated the twodimensional data (m/z-compounds) for the chemical compounds from all the herbs from HSYFD. Second, compounds were characterized by the systematically matched information in Agilent MassHunter Qualitative Analysis 10.0 software by the function "find by formula". An average mass error of   Table 1. A total of 108 chromatographic peaks were identified, of which 65 compositions were found in the positive mode, 37 compositions were detected in the negative mode, and 6 compositions were found in both the positive and negative modes.
The structural types of these compounds were mainly flavonoids, sesquiterpenoids, amino acids, and phenylpropanoids. Among the constituents, 9 compounds were from AR, 13 compounds were from CRP, 17 compounds were from MOC, 2 compounds were from TF, 12 compounds were from EH, 16 compounds were from NRR, 6 compounds were from ZRR, 12 compounds were from AS, and 8 compounds were from PH. 13 compounds were common compositions of multiple medicinal materials, such as both AS and EH contain ferulic acid.

Analysis of Prototype Constituents of HSYFD in Rat Serum.
As 108 compounds were found from database 1.0, the retention times of those compounds were added in the library list, and therefore generated the three-dimensional data set (retention time-m/z-compounds) in Personal Compound Database Library (PCDL), which certainly was the database 2.0. 14−16 Normally, the method criterion for retention time was 0.25 min, and the average mass error of adduct and isotopes was 10 ppm. The rat serum was analyzed by the same UHPLC−Q-TOF-MS method, and therefore the absorbed prototype compounds were screened out by quick-matching the peaks in the database 2.0 using the Agilent MassHunter Qualitative Analysis 10.0 software.
Eleven chromatographic peaks were identified (Table 2, Figure 3). Among the constituents, hinesol was from AR, 10gingerol and 6-gingerol were from ZRR, scopoletin was from NRR, anonaine was from MOC, loliolide and kumatakenin were from PH, norephedrine was from EH, histidine was from AS, p-coumaric acid was from MOC, CRP, and NRR, and 4hydroxybenzoic acid was from TF and AS.

Network Pharmacology Analysis. 2.4.1. Potential Targets of the Active Ingredient of HSYFD.
A total of 422 putative targets of the 11 identified prototype constituents of HSYFD were obtained from the databases, and 704 potential targets were obtained by searching the genes related to Han-Shi-Yu-Fei symptoms of COVID-19 in the TCMIP V2.0. There were 48 overlapping targets from the compound-related targets and Han-Shi-Yu-Fei-related targets ( Figure 4A). By constructing a protein−protein interaction (PPI) network for the abovementioned targets, it was found that a total of 42 targets interacted, forming a complex interlaced network. Among them, the betweenness centrality, closeness centrality, and degree of EGFR, TP53, TNF, JAK2, NR3C1, TH, COMT, and DRD2 were all higher than the average and were considered to be the core targets ( Figure 4B, Table 3). To establish the relationship of herbs, compounds, and targets, we constructed a network of them. As shown in Figure 4C, the network consisted of 61 nodes and 84 edges, and the active ingredients of HSYFD correspond to multiple targets, and one target also corresponds to multiple active compounds; among them, 10-gingerol corresponds to 17 targets, 6-gingerol corresponds to 15 targets, both kumatakenin and anonaine correspond to 10 targets, they were the components with the most associated targets.

GO and KEGG Analyses of Potential Targets of the Active Ingredient of HSYFD.
To probe the mechanisms of HSYFD in the treatment of mild stage of COVID-19, 42 targets were enriched by GO functional enrichment and KEGG pathway enrichment analyses. A total of 988 GO teams with a p-value < 0.001 were enriched, including 851 biological processes, 64 molecular functions, and 73 cellular components ( Figure 4D). In detail, the biological process category mainly involved cellular process, biological regulation, and response to stimuli. Molecular function enrichment results primarily included binding, catalytic activity, and molecular transducer activity. In the cellular component category, targets were considered to be enriched in cell, cell part, and membrane. Multisteps were reported in the SARS-CoV-2 entry process. Specifically, in the steps that fusion between viral and cellular membranes, the viral RNA was released into the host cell cytoplasm for uncoating and replication. 17 GO analysis suggested that the therapeutic effect of HSYFD on COVID-19 may be attributed to its disruption of the interaction between viral and host cellular membranes.
Similarly, KEGG signaling pathways (P < 0.01) included multiple pathways that had been shown to be associated with COVID-19 (Table 4). For instance, both MAPK and PI3K/ AKT signaling pathways contain multiple core targets (TNF, TP53, EGFR, and JAK2). It was reported that the MAPK pathway could regulate many cellular processes and were essential for immune cell function, 18 and the PI3K/AKT signaling pathway was associated with the activation of both CD147 and furin, which was also involved in the endocytosis of SARS-CoV-2. 19 Additionally, the process of virus invading cells was reported to cause changes in erbB, HIF-1, and mTOR pathways by the integrative proteo-transcriptomics analysis of Huh7 cells infected with SARS-CoV-2. 20 Moreover, SARS-CoV-2 could trigger inflammation through the JAK-STAT pathway, leading to the development of cytokine storms in cells such as lung cells and endothelial cells, causing lung injury. 21 2.5. Molecular Docking Results. The binding activities of these core targets and the core compounds were preliminarily verified by molecular docking technology. 22 Results showed that the binding energies of almost all core targets to the molecules less than −5.0 kcal/mol ( Figure 5). For example, the binding energies of COMT, JAK2, NR3C1, and TP53 to 6gingerol were −5.8, −6.6, −6.7, and −5.8 kcal/mol, indicating that they displayed good binding activities, which was largely

DISCUSSION
For orally administrated TCM recipe, systematic identification of the blood-absorbing compounds is critical to understanding its pharmacodynamic material basis and the therapeutic mechanism. 23 Therefore, first, a total of 108 components were identified from HSYFD by the UHPLC−Q-TOF-MS technique. The qualitative analysis characterization compounds mainly consisted of flavonoids, sesquiterpenoids, amino acids, and phenylpropanoids. Next, a total of 11 prototype components were identified from the rat serum after oral administration of HSYFD, which could be recognized as the potential material basis for the efficacy of HSYFD. In particular, kumatakenin from PH showed good inhibitory activity against SARS-CoV-2 replication in Vero E6 and Calu-3 cells, whose inhibitory effects were even better than those of lopinavir/ritonavir and chloroquine. 24 In addition, 10-gingerol and 6-gingerol, two key compounds based on degree ranking in herb−constituent−target network analysis, showed antiinflammatory, immune regulation, and anti-nausea activities. 25 In some situations, the elevated inflammatory markers were also identified as risk factors for psychoticism symptoms, which could induce depression in COVID-19 patients. 26,27 Moreover, scopoletin from NRR promotes glutamatergic modulating effects and could regulate depression by increasing synaptic plasticity in prefrontal regions. 28 Although COVID-19 was diagnosed based on reverse transcription polymerase chain reaction, the patients were classified as mild, moderate, severe, or critical depending on the clinical severity of their prominent clinical symptoms. In the initial viral phase, about 80% of patients usually have mild or asymptomatic disease. In the second phase, host−virus interactions take place which dictate the outcome for the subsequent phases of the disease, which corresponded to a hyper-responsiveness of the immune system. Given this characterization, the typical clinical manifestations were the representatives of the targets of COVID-19. Thus, it is worth noting that the disease targets were obtained by the clinical symptom-guided searching, and, interestingly, the commonly used known related targets for COVID-19 such as NR3C1, 29 JAK2, 30 JAK3, 31 MIF, 32 and STAT3 33 had been overlapped.
KEGG analysis revealed many pathways associated with COVID-19, such as MAPK signaling pathway, PI3K/AKT signaling pathway, and JAK-STAT pathway. The PPI network found that EGFR, TP53, TNF, JAK2, NR3C1, TH, COMT, and DRD2 were the core targets of HSYFD in the treatment of COVID-19. The role of inflammatory mechanism had been highlighted in the pathogenesis of COVID-19. Tumor necrosis factor (TNF), another key mediator of triggering the cytokine storm, showed high levels in organs such as the lymph nodes of COVID-19 patients, which could prevent the body from producing memory B cells, thereby inhibiting the generation of a durable immune response. 34 At present, the JAK inhibitor baricitinib, which could reduce inflammation by inhibiting the levels of cytokines such as IL-6, IL-1β, and TNF-α and regulate the immune environment, was approved by FDA for the treatment of hospitalized adult patients with COVID-19. 35 According to the COVID-19 pathological characteristics, the overexpressed EGFR in the infected lung cells exacerbates pulmonary fibrosis as the severe consequence of cytokine storm, evidenced by the transcriptomic data of patients. 36 Additionally, the lung lesions in COVID-19 patients could be alleviated by the treatment of anti-EGFR antibodies like nimotuzumab, accompanied with the reduced plasma IL-6 level. 37 In addition, protective and immunomodulatory effects of TCM were observed by augmenting TNF-α and IL-6 expression in the animal model of acute lung inflammation. Recent accumulating data suggested that TP53 gene therapy may be a new treatment for COVID-19 patients, 38 and it was therefore concluded that the compounds from HSYFD might

ACS Omega
http://pubs.acs.org/journal/acsodf Article augment TP53 expression. For example, 6-gingerol, which showed good binding activities to TP53, could promote the activation of TP53. 39 The subsequent molecular docking computational analysis confirmed the constituent−target interactions from the network pharmacology results. Based on the description above, it was speculated that HSYFD may play a role in the treatment of COVID-19 via MAPK signaling pathway, PI3K/ AKT signaling pathway, and JAK-STAT pathway.
Although 108 compounds were identified for HSYFD, only 11 prototype compounds as the absorbed constituents were characterized in the rat serum. However, those unabsorbed constituents were not considered key active constituents because of the multiple complex ingredient−body interactions. Sometimes, unabsorbed compounds such as flavonoids were metabolized by the gut microbiota to smaller metabolites, which were more bioavailable than their precursors. 40 Moreover, microbiological studies showed that unabsorbed components could prompt the growth of beneficial bacteria and inhibit the growth of disease-causing bacteria, contributing to maintain the homeostasis of the digestive system and gut flora. 41 Of course, the alterations of gut microbiota in COVID-19 patients consequently may lead to the development of gut dysbiosis-related diseases. Therefore, it was also recommended for the recovery of gut microbiota balance while treating COVID-19 patients. 42 Many Chinese herbs and preparations have demonstrated properties of restoring intestinal barrier function and intestinal homeostasis, which may further modulate the immune function after SARS-CoV-2 infection. 43,44 Understanding the role of compounds in maintaining intestinal flora balance could provide new ideas for preventing and treating COVID-19.
At present, FDA-approved anti-SARS-CoV-2 drugs such as Paxlovid, Molnupiravir, and remdesivir, mainly focus only on individual targets like 3CL protease and RdRp, and it took plenty of time and capital to discover the new drug, which is still effective on the front of continuous mutation of SARS-CoV-2. 45−47 Based on a specific theory, numerous clinical data evidenced the TCM for many known infectious disease treatments. More importantly, TCM played an irreplaceable role in the prevention and treatment of unknown COVID-19 since its first epidemic, especially in the treatment of mild patients. 48,49 Furthermore, current clinical trials are devoted to finding the superiority of the combination of the TCM and western medicine in the treatment of COVID-19 patients infected with Delta or Omicron variants, when compared with the western medicine treatment. 50

CONCLUSIONS
In this work, the chemical components of HSYFD in vivo and in vitro were analyzed by a UHPLC−Q-TOF-MS method, and the mechanism for treating COVID-19 was explored by clinical symptom-guided network pharmacology technology with molecular docking. A total of 108 components were identified from HSYFD, of which 11 components were detected in the rat serum after its oral administration. MAPK signaling pathway, PI3K/AKT signaling pathway, and JAK-STAT pathway were recognized as the key pathways of HSYFD for confronting COVID-19, with COMT, DRD2, EGFR, JAK2, NR3C1, TNF, and TP53 as the key targets. Our work also verified the feasibility of clinical symptom-guided network pharmacology analysis for the chemical compounds, which provided a possible agreement between the points of view of TCM and western medicine on the disease. Hesperidin (P06D9F77001), 6-shogaol (P06N9L74388), and notopterol (R28m9f57295) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (purity ≥ 98%). Ultra-pure water was prepared using a Milli-Q water purification system (Bedford, France). Acetonitrile and methanol were of UPLC grade (Merck, Darmstadt, Germany). All other reagents were of analytical grade.

Preparation of the HSYFD Extract.
AR (105 g), CRP (70 g), MOC (70 g), PH (70 g), TF (42 g), EH (42 g), NRR (70 g), ZRR(70 g), and AS (70 g) were co-decocted in water twice, for 1 h each time. Water added in the first round was 10 times the total weight of the herbs, and in the second round it was 8 times the total weight of the herbs. The extraction solutions were then filtered, combined, and concentrated under vacuum to yield 610 mL of a concentrated decoction (HSYFD, 0.998 g/mL). About 10 mL of the HSYFD was centrifuged at 3000 r/min for 10 min, transferred into a 1.5 mL centrifuge tube, and centrifuged again at 13,000 rpm for 15 min. 200 μL of the supernatant was then subjected to UHPLC−Q-TOF-MS analysis. The remaining decoction was freeze-dried to obtain lyophilized powder, dissolved in normal saline by ultrasound to form a solution equivalent to a crude drug concentration of 7 g/mL, and stored at 4°C before intragastric administration.
Parameters for Electrospray Mass Spectrometry (ESI-MS) analysis were set as follows: scan range, mass-to-charge ratio (m/z), 100−1700; dry gas (N 2 ) temperature, 350°C; dry gas flow, 11.0 L/min; atomizer gas pressure, 45 psi; capillary voltage, 4000 V (positive)/3500 V (negative); divider voltage, 140 V; collision energy, 30 V. All data were obtained with reference masses at m/z 121.0509 and 922.0098 in the positive mode, and at m/z 1033.9881 in the negative mode. QC samples were injected after every five samples during the sequence analysis to evaluate the analytical performance.

Animals and Drug Administration.
Twelve SPF grade male Sprague−Dawley rats (220 ± 20 g) were obtained from the Shanghai Institute of Planned Parenthood Research (Shanghai, China) and were housed in accordance with the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM201113012). All rats were stored in a room with controlled temperature (22−26°C ) and humidity (40−70%), under a 12 h light−dark cycle. Animals were acclimatized to the facility for 1 week before the start of the experiment. Rats were randomly divided into two groups (n = 6 for each group) before the experiment. Rats in the HSYFD group were dosed at 70 g crude drug/kg/day for three consecutive days, while 0.9% normal saline (2 mL/day) was administered to the rats in the control group.

Serum Sample Collection and Pretreatment
Method. Blood samples were collected from the infraorbital vein 1 h after the last administration of HSYFD and stored in 5 mL BD Vacutainer Plus Plastic Blood Collection Tubes. Serum was obtained by centrifugation at 3000 rpm at 4°C for 10 min after standing for 2 h at room temperature (25°C), and 100 μL of which was deproteinized with 400 μL of ice-cold methanol. After being vortexed for 1 min and centrifuged at 4°C at 12,000 rpm for 15 min, the supernatant was transferred to a new centrifuge tube, lyophilized, and then reconstituted in 100 μL of methanol. The supernatant was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was prepared for UHPLC−Q-TOF-MS analysis. 5.6. Target Network Analysis. The potential targets of the compounds were obtained from Swiss Target Prediction (http://www.swisstargetprediction.ch) and TCMSP (https:// tcmspw.com/tcmsp.php). Based on TCMIP V2.0 (http:// www.tcmip.cn), the HSYF phenotype targets were obtained by retrieving the entries of clinical symptom of mild stage of COVID-19, including fever, cough, abnormality of the pharynx, fatigue, anergy, dyspnea, abnormality of the stomach, nausea, vomiting, diarrhea, abnormality of the tongue, and abnormality of the vasculature.
The ImageGP website (http://www.ehbio.com/ImageGP/) was used to identify the common targets of HSYFD and COVID-19. The OmicShare cloud platform (https://www. omicshare.com/tools/) was used for dynamic GO enrichment analysis and KEGG pathway enrichment analysis of the abovementioned common targets, and "homo sapiens" was selected as the gene source. Significance was set at P < 0.001 for GO entries and at P < 0.01 for signal pathways. The common targets were also used to construct a PPI network model on the String website, and then input into Cytoscape 3.7.2 to obtain the PPI network, and the network analyzer function in the software was used to obtain the relevant network topology parameters. The core targets of HSYFD treatment of COVID-19 were screened with the three indices of degree of degrees, betweenness centrality, and closeness centrality, all of which exceeded the average values. 51 5.7. Molecular Docking Analysis. The 3D structures of the core targets were obtained from the PDB database (http:// www.rcsb.org) and AlphaFold (https://alphafold.ebi.ac.uk). PyMOL software was used to remove excess water molecules and inactive ligands. 3D structures of the compound corresponding to the core target from PubChem were converted to PDB format by openbabel 3.1.1 and saved as pdbqt format using autodock tools 1.5.7. Autodock Vina and PyMOL software were used for molecular docking and visualization. 52 pharmacology, G.-Y.J., H.-Q.W., and S.-H. P.; project administration, W.-S.C.; funding acquisition, F.Z. and W.-S.C. All authors have read and approved the final version of the manuscript.

Notes
The authors declare no competing financial interest. The animal study was reviewed and approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM201113012). The raw data sets used and analyzed during the study are available on request to the senior author (W.-S.C., chenwansheng@smmu.edu.cn).