Recently, a new type of coronavirus disease 2019 (COVID- 19) , with rapid spread and high morbidity, broke out worldwide. Fortunately, a number of endeavors revealed that traditional Chinese medicine (TCM) treatment could effectively relieve symptoms and prevent the fatal deterioration of this disease [1] [2] [3] [4] . As a typical TCM formulation, Lianhuaqingwen (LHQW) capsule has been confirmed to show therapeutic effects by clinical research and observation 5, 6 . More importantly, in April 2020, the "Pharmaceutical Supplement Application Document" issued by National Medical Products the symptoms of fever and stagnation of the lung 9 . An anti-influenza A (H1N1) trial demonstrated that LHQW had better effect on the alleviation of fever, cough, sore throat, and fatigue, in comparison with oseltamivir. Furthermore, it showed comparative therapeutic effectiveness in reduction of illness duration and viral shedding duration 8, 10 . Although molecular targets of LHQW remain unknown, pharmacological activities of LHQW have been explored in vitro. Ding et al. 7 reported that the raw material of LHQW (dissolved in DMSO as stock solution) inhibited the proliferation of influenza viruses from various strains in vitro, with the 50% inhibitory concentration (IC 50 ) ranging from 0.35 to 2 mg/mL. And it could also suppress virus-induced nuclear factor κB (NF-κB) activation and alleviate virus-induced gene expression of interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), interferon-γ-inducible protein-10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1). Using a similar in vitro approach, it was demonstrated that LHQW exerted anti-SARS-CoV-2 activity via inhibiting virus replication and reducing cytokine release from host cells 5 . Furthermore, networking analysis has been applied to identify main effective components in raw LHQW materials. For example, Wang et al. 11 revealed 15 active components of LHQW and 61 related targets by using network pharmacology. Furthermore, You et al. 12 found that the active components of LHQW might inhibit cytokine storm by regulating various inflammatory signal pathways by network pharmacology analysis. Just like a Western medicine product, it is well recognized that the efficacy and safety of TCM are associated with the chemical constituents of TCM including prototype components and their metabolites in the circulation, which are directly associated with the whole process of absorption, distribution, metabolism, and excretion (ADME) 13, 14 . Identification of TCM components in animals or human circulation followed by activity testing of these components is another approach to study molecular mechanisms of pharmacological effects of TCM. For example, Wang et al. 15 combined serum pharmacochemistry-based screening and high-resolution metabolomics analysis to find bioactive components. However, information on exposure, metabolism and disposition of LHQW in animals or human is not available in the literatures.
In addition to directly testing extracted raw materials from a TCM product or virtual network pharmacology analysis, phytochemical separation of individual components followed by pharmacological activity testing in vitro is a more common and useful practice in searching for active constitutes of an effective herbal medicine product 16 . However, this approach may not be able to focus on TCM components that are either absorbed in and exposed to human after oral administration or formed in human via biotransformation by metabolizing enzymes in human.
The first primary objective of this study was to determine the component profiles of LHQW in J o u r n a l P r e -p r o o f human plasma and urine, after oral administration of multiple therapeutic doses to human subjects.
The LHQW formula was composed by 13 18 , metabolomics approach has been increasingly used for untargeted detection of TCM components 14 . In this study, we applied a previously developed analytical strategy for studying ADME of TCM in vivo 19, 20 , which used precise-and-thorough background subtraction (PATBS) for sensitive and comprehensive detection and structural characterization of TCM components in complex biological samples.
The second primary objective of this study was to identify potential anti-COVID-19
pharmacological components of LHQW, that were exposure to human after multiple oral doses.
SARS-CoV-2 was reported to achieve human infections through the binding of its spike protein (S protein) to angiotensin converting enzyme 2 (ACE2) as receptor 21 , which is a negative regulator with the ability to maintain the steady state of renin-angiotensin system (RAS) and is critical to the physiology or pathology of all organs 22 . ACE2 has been found to be widely distributed in the heart, kidney, testis, adipose tissue, brain tissue, vascular smooth muscle cells, and gastrointestinal tract 23 .
Zhao et al. 24 applied high-throughput single-cell sequencing analysis technique to study 43,134 lung cells and found that ACE2 was expressed in 0.64% lung cells, and in 1.4% alveolar type II cells (AT2), which became the binding targets of SARS-CoV-2 as an invasion starting point. Furthermore, Xu et al. 25 confirmed that ACE2 is a viral receptor by studying the binding capacity of the SARS-CoV-2 protein crystal structure to human ACE2 receptor. In general, the above studies J o u r n a l P r e -p r o o f indicated that screening drugs with ACE2 targeting ability are promising for the treatment of patients with SARS-CoV-2 infection. Hence, comprehensive 2D ACE2 biochromatography system was established for efficiently screening for active LHQW components exposed to human after repeated oral does. The ACE2 inhibitory activity of hits from the biochromatography screening was further confirmed by using the pharmacodynamics study of surface plasmon resonance (SPR) and analysis using ACE2 inhibitory activity assay kit as well as supported by results from docking simulation. nitrogen. Reconstitutes were obtained by re-dissolving the residues using 100 µL 70% methanol.
After 10 s of ultrasonication, vortex mixing, and 12,000 rpm centrifugation (D3024, DLAB Scientific Co., Beijing, China) for 5 min, the supernatants were collected for further analysis. Before administration, blank plasma samples were prepared in a similar manner.
The urine samples from 6 subjects (subject numbers 1, 3, 6, 8, 9, and 12) on Day 8 were prepared as follows. The urine samples from subjects collected in the periods of 0-4, 4-10, 10-24, 24-32, or 32-48 h after drug dosing (100 µL urine at each time point) were mixed to obtain a 3-mL sample. Furthermore, the samples were respectively enriched and purified by Oasis ® HLB 3cc (60 mg) extraction cartridges. The methanol eluent was collected and dried with nitrogen at 37 °C.
Reconstitutes were obtained by re-dissolving the residues using 100 µL 70% methanol. After 10 s of ultrasonication, vortex mixing, and 12,000 rpm centrifugation (D3024, DLAB Scientific Co., Beijing, China) for 5 min, the supernatants were collected for further analysis. Before administration, blank urine samples were prepared in a similar manner.
To determine component profiles of LHQW in human body, human plasma and urine samples were High resolution full-scan MS and MS 2 data were collected at resolving power of 35,000 and 17,500, respectively. Stepped HCD collision energy of 35% was used. The temperature of capillary was set at 320 °C, and the spray voltage was 3.5 kV for negative ion mode, and 3.8 kV for positive ion mode.
The flow rate of sheath gas (N 2 ) and auxiliary gas (N 2 ) were 40 and 10 arb, respectively. The data obtained were analyzed by Thermo Xcalibur 3.0.63 Qual Browser (Thermo Fisher Scientific).
After collection of MS and MS/MS data of the LHQW test solution, structural characterization and confirmation of LHQW constituents were carried out by using Compound Discoverer 3.1 software (Thermo Fisher Scientific). The main parameters of this software were set as follows: align retention times, maximum shift 2 min; mass tolerance, 5 ppm; detect compound, mass tolerance 5 ppm; intensity tolerance, 30%; minimal peak intensity, 500,000; data sources, mzCloud, mzVault, MassList, and ChemSpider Search; and S/N threshold, 3, after which the compounds searching and matching were conducted. And then, the software searching report was combined with the fragmentation rule of reference MS as well as the corresponding characteristic fragment ion information. In addition, 34 reference standards were used for structural confirmation.
Detection and structural characterization of LHQW components in human plasma and urine were carried out by a LC-HRMS workflow, which was previously developed for ADME study of a J o u r n a l P r e -p r o o f TCM product in vivo 19 . Briefly, the workflow used PATBS, an untargeted data-mining technology, to detect LHQW components in a plasma or urine sample. Once the LHQW components were found in full scan LC-HRMS dataset, their MS/MS spectral data were either retrieved from the MS/MS datasets acquired by ddMS 2 methods or acquired via targeted production ion scanning analysis. And then, structural conformation of prototype components of LHQW in the plasma and urine samples was accomplished by comparing their LC-HRMS data with LHQW test solution. Meanwhile, metabolites of LHQW components were structurally characterized using some functions of Compound Discoverer 3.1 software, including biotransformation matching, spectral similarity comparison and fragmentation interpretation.
The reaction scheme was shown in Fig 
The comprehensive 2D ACE2 column/C18 column/TOFMS system was performed on an Agilent 1200 series HPLC system coupled with a 6220 TOF mass spectrometry consisting of an auto-sampler, which was controlled by Agilent MassHunter Workstation (Agilent Technologies, Palo Alto, CA, USA DMSO from 0.024 to 50 μmol/L, and was injected into the reference channel and ACE2 protein channel, respectively, at a flow rate of 30 μL/min. The coupling and dissociation time were both 120 s. Biacore T200 evaluation software was used to fit the affinity curves by the steady-state affinity model (1:1), and the equilibrium dissociation constant (K D ) was calculated.
The ACE2 inhibitory activities of the candidate active components were tested by ACE2 Inhibitor was employed for docking the compounds to the binding site of protein and the best docking pose of the compound was kept based upon Glide scoring function (G-score).
To facilitate the identification of LHQW components in human plasma and urine, LHQW test J o u r n a l P r e -p r o o f solution was first analyzed by the UHPLC-HRMS. As shown in Fig. 2 Fig. S2 ).
Insert Fig. 2 
An unprocessed chromatographic profile of a human urine sample after LHQW administration (Fig.   3B) shows that a majority of peaks were endogenous components as those in a control urine sample LC-HRMS data with those of LHQW test solution (Fig. 2) , reference standards, as well as spectral interpterion and biotransformation marching. As a result, 70 LHQW components in the human urine were tentatively identified or confirmed (Table S1 ). (Table S1 ). In addition to detection and structural characterization, the UPLC-HRMS profiles provides semi-quantitative information on the LHQW components, while LC-UV profile of the plasma does not exhibit a majority of these LHQW components due to low concentration in human plasma. Based on the results of analyzing urine (Supporting Information Fig.   S3 ) and plasma samples, 87 significant LHQW prototype components were absorbed after repeated oral administration, and 45 LHQW metabolite components were formed in humans (Table S1 ). Thus, we considered that a total of 132 LHQW related components were exposed to human after repeated oral administration of LHQW.
Insert Fig. 4 J o u r n a l P r e -p r o o f
MPTS and GMBS were applied for biochromatographic stationary phase modification, in order to achieve covalent immobilization of ACE2 recombinant protein for screening potential anti-COVID-19 components from LHQW. Using this strategy, the interaction yield of mercapto with the maleamide group was high under mild reaction conditions and the self-polymerization could be avoided 26 . The exposed N-hydroxysuccinimide groups were practical linkages for amine coupling of target proteins with high recovery and stable chemical environment 29 . As shown in Fig. 1B, ACE2 antibody-based immunoblotting shows that the immobilized ACE2 could be digested by 0.3% pronase from the ACE2 biochromatographic stationary phase, indicating the successful covalent immobilization of ACE2. The content of immobilized membrane protein on 40 mg MPTS/GMBS modified silica gel was measured as 18.24±1.05 μg, while a good protein recovery of >90% was achieved.
As shown in Fig. S1A , the first fraction from the ACE2 biochromatography column was enriched into a 500 μL sample loop 1 at the 10-port valve switched at position 1. After switching to position 2 ( Fig. S1B) , the enriched components in sample loop 1 were pumped into an Agilent Poroshell EC-C18 column and TOFMS detector, for further separation and qualitative analysis of chemical components. Meanwhile, the second fraction of ACE2 column was pumped into sample loop 2 and waited for analysis, with the alternate switching of 10-port valve. In order to get comprehensive 2D biochromatographic analysis results, it was necessary to collect at least two fractions across a retention peak. Hence, the collection period was set at 2.5 min for each round of analysis. This novel comprehensive 2D ACE2 column/C18 column/TOFMS system realizes the automated and high-throughput analytical process, rapid separation, and accurate identification, which are helpful in discovering potential ACE2 target ligands from complex chemical samples.
The comprehensive 2D ACE2 column/C18 column/TOFMS system was applied into screening potential ACE2 target components from enriched urine sample and extract of LHQW. As shown in retention components were screened out after deducting the blank urine background (Fig. 5B ). More importantly, the complex components from the extract could also be characterized by the 2D spectrum, obtained from the comprehensive 2D ACE2 biochromatography system (Fig. 5C ). In details, these prototype components and their metabolites were tentatively identified by TOFMS J o u r n a l P r e -p r o o f according to the "generate formula from mass peak" function of Agilent MassHunter Qualitative Analysis software and the data of fragment ions. As shown in Supporting Information Furthermore, rutin and its deglycosylated compounds (quercetin) had also been proved to show certain binding ability with ACE2. The sensorgrams of another five components with affinities to ACE2 protein were shown in Supporting Information Fig. S4A-S4E . According to the general criteria 31 , the situation of R max value exceeding 2 times of the response of compounds should be considered as nonspecific binding ligands. As shown in Table 1 Insert Table 1 and Fig. 6 
The effects of positive drug and all candidate components on the activity of ACE2 were tested using the ACE2 Inhibitor Screening Kit. Based on the SPR results, rhubarb anthraquinone compounds, forsythiaside compounds, neochlorogenic acid and its isomers, as well as prunasin and glycyrrhizin with high in vivo exposure levels were selected. As shown in Fig. 7A Radix et Rhizoma did not show significant inhibitory effects (Fig. 7C) . Moreover, as shown in Fig.   7D , three chlorogenic acid isomers all reveal moderate ACE2 inhibitory activities with IC 50 at about 40 μmol/L, while the two components prunasin and glycyrrhizin, with strong ACE2 binding affinities, did not show significant peptidase inhibitory effects. They failed to bind with any of the peptidase active site, which was confirmed by molecular docking assays. It is worth mentioning that the in vivo exposure concentration of rhein is relatively high, with good activity, indicating that it might be the main active ingredient of LHQW to exert ACE2 inhibition ability. Other active compounds, such as prunasin and glycyrrhizin, had high in vivo exposure concentrations but low inhibitory activity; while forsythosides A and I, or neochlorogenic acid and its isomers had good in vitro activity, but low in vivo exposure, which might show synergistic effects with rhein. This might also reflect the combined synergistic effect of TCM.
Insert Fig. 7 
Docking simulation studies were carried out to investigate the binding sites of six representative components. Previous studies showed that the main interaction interface of ACE2 that binding with the RBD region of S protein are Gln24 to Tyr83 and Gln325 to Arg393 32,33 . It is worth mentioning that the peptidyl dipeptidase activity sites were widely distributed in various regions at the N-terminal of ACE2 protein, including Trp271 to Arg273, His345 to Ala387, and His505 to Arg514 34, 35 . The potential intersection region of peptidase activity and S protein binding is around His345 to Arg393. Thus, the compounds that block S protein binding to ACE2 may not have strong catalytic activity to inhibit ACE2 peptidase 36 . Recent reports showed that some potential ACE2 inhibitors screened by computer simulations do not directly bind to the interface between ACE2 and spike protein complex 37 , indicating that these compounds were not suitable for inhibiting SARS-CoV-2 infection. In this case, it may be worthy to conduct further exploration to identify whether their specific binding site is directly bound to the contact surface between ACE2 and spike complex or ACE2 peptidase active sites, as this interaction might determine the compound's molecular mechanism in inhibiting SARS-CoV-2 infection 21 Glycyrrhizin also showed good affinity to ACE2 protein but poor inhibitory effect. The docking result indicates glycyrrhizin could only bind to the region (Lys26 to Asp30) and not bind to any of the peptidase region. Rhein showed the best ACE2 inhibitory effect, and was confirmed by the molecular docking that the carboxyl could form hydrogen bond to Ala387, a key peptidase active site.
The similar situation was observed on emodin 8-O-β-D-glucoside. However, aloe-emodin showed good affinity to ACE2 protein but no inhibitory effect. The docking result reveals it could bind to the sites of Glu35 and Gln76 with phenolic hydroxyl group, not peptidase sites (Supporting Information   Fig. S5 ). Neochlogrogenic acid and its isomers could form 4 hydrogen bonds to the intersection region of peptidase activity and S protein binding (Ala348 to Arg393), indicating the potential double inhibition of S protein binding and ACE2 enzyme activity.
Insert Fig. 8 
A TCM product usually contains from several dozens to a few hundreds of prototype components, while each constituent might change quantitatively with environmental factors or processing conditions 14, 38 . TCM component profiles in human and animals become much complicated due to biotransformation by metabolizing enzymes, which makes identification of in vivo TCM metabolite components and their formation pathways very challenging.
A novel strategy for the discovery of ACE2 inhibitory active components in LHQW that may play important roles in COVID-19 pneumonia treatment was established in this study (Fig. 9 ). In the first step, UPLC-HRMS was utilized to comprehensively analyze and identify the chemical compositions of LHQW. Resultant information was utilized for subsequent identification of LHQW components exposed to human. In the second step, major LHQW components in human urine were detected and structurally characterized followed by their inhibitory effects on ACE2 using 2D biochromatography.
In the last step, SPR and ACE2 inhibitory activity assay were applied to confirm the pharmacodynamics of active components from the screening, while computer docking was used to elaborate on the possible sites of pharmacology action. The most important element of this strategy was to combine in human exposure study with 2D-biochromatography analysis, which was proved to be efficient in revealing the active components from LHQW that may exhibit inhibitory effects on J o u r n a l P r e -p r o o f ACE2 in human.
In this study, a LHQW test solution sample was initially used for setting up for UPLC-HRMS method as well as characterizing structures of LHQW prototype components (Fig. 2) to support identification of LHQW component in vivo. As shown in Table S1 , 126 prototypes in the LHQW test solution were tentatively identified and 34 prototypes of them were fully confirmed by using reference standards, which were significantly more than the total prototype components found in LHQW in a previous study although major components identified from the two studies are similar 17 .
Furthermore, the HRMS-based PATBS technique was applied to detection of LHQW components in human urine and plasma samples using a workflow previously developed for studying ADME of TCM in vivo 19 . PATBS is an untargeted data-mining tool capable of finding TCM components or other types of xenobiotics in a test in vivo sample regardless of their molecular weights, mass defects, isotope patterns and fragmentations since these xenobiotics were not present in a corresponding control sample such as a pre-dosing plasma and urine sample. As shown in Fig. 3 , PATBS not only removed large portions of endogenous components and background noise to reveal minor LHQW components ( Fig. 3D and E) , but also significantly cleaned full-scan MS spectral data to make molecular ions of interest as dominant ion species (Fig. 3F ). Both metabolomics approach and PATBS are untargeted data mining tools and their effectiveness of finding xenobiotics is comparable.
However, PATBS has two significant advantages 39 . First, PATBS can be used in a combination with targeted or semi-targeted data mining tools, such as extracted ion chromatographic processing or mass defect filters, to increase detection sensitivity and selectivity 20 . Second, PATBS is capable of processing a single test sample by using a single control sample, while the metabolomics approach requires analysis two groups of test and control samples and each of the group has three or more samples. Results from this study demonstrate that HRMS combined with PATBS is a superior data-mining tool for studying ADME of a TCM product in human.
A good understanding of human exposure to TCM components after oral administration of a TCM product is considered as a very important step to investigate molecular mechanisms of its pharmaceutical effects in human. In this study, we detected and characterize a total of 107 LHQW components (66 prototypes and 41 metabolites) in human plasma after oral administration of therapeutic doses (Table S1 ). In addition, UPLC-HRMS profiles of plasma samples provided semi-quantitative estimation on the LHQW components in the human circulation ( Fig. 4A and B ).
Based on the information, more accurate quantification of circulating components in human can be performed using LC-MS and their reference standards. Furthermore, we found additional 21 LHQW prototype components that were absorbed and then underwent rapid metabolic clearance or renal J o u r n a l P r e -p r o o f excretion. These LHQW components were also exposed to human even without the significant presence in the circulation. The approach significantly reduced cost and time in studying pharmacological mechanism of LHQW by avoiding testing over a large number of individual prototype components that were found in LHQW test solution, but unlikely exposed to human.
The experimental results from this study show that many of the main exposed constituents of [40] [41] [42] [43] . It is worth mentioned that the current studies on the combination of the main exposed components of LHQW in the body and its possible interaction with ACE2 have been rarely reported. This interesting result also illustrates that it is necessary to conduct research on the efficacy of TCM from an in vivo exposure perspective.
In It is worth mentioning that these constituents not only showed good affinity to ACE2 but also could effectively bind to the contact surface of ACE2 and spike complex, which was confirmed by computer-aided docking results. To the best of our knowledge, this was the first report of comprehensive study on human exposure to LHQW components. More importantly, it was found that several LHQW components exposed to human might play potential roles in inhibiting SARS-CoV-2 by significantly affecting the binding between ACE2 and S protein, which is an important route of preventing virus infection. This study provided direct chemical and biochemical evidences associated with molecular mechanisms of clinical use of LHQW for prevention and treatment of COVID-19. In addition, this study demonstrates the utility of human exposure-based approach to identifying pharmacologically active components in a herbal medicine product with approved therapeutic effects. J o u r n a l P r e -p r o o f 