Comprehensive Proling and Characterization of The Absorbed Components and Metabolites in Mice Serum and Tissues Following Oral Administration of Qingfei Paidu Decoction by UHPLC-Q-Exactive-Orbitrap HRMS

Background: Qingfei decoction (QFPDD) is a Chinese compound formula recommended for combating 2019 (COVID-19) by National Health Commission of the People's Republic of China. This study aims to identify the main constituents in QFPDD and the absorbed components (including prototypes and metabolites) in serum and tissues after oral administration of QFPDD to mice. Methods: A practical and sensitive method of UHPLC-Q-Exactive-Orbitrap HRMS was developed to identify the chemical constituents in QFPDD and the absorbed prototypes as well as the metabolites in mice serum and tissues following oral administration of QFPDD. Results: A total of 405 chemicals, including 40 kinds of alkaloids, 162 kinds of avonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds in the water extract of QFPDD were tentatively identied via comparison with the retention times and MS/MS spectra of the standards or refereed by literature. With the help of the standards and in vitro metabolites, 195 chemical components (including 104 prototypes and 91 metabolites) were identied in mice serum after oral administration of QFPDD. In addition, 165, 177, 112, 120, 44, 53 constituents were identied in the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. Conclusions: An UHPLC-Q-Orbitrap HRMS based method was established for chemical proling the constituents in QFPDD, while the absorbed prototypes and metabolites occurring in mice serum and tissues were investigated following oral administration of QFPDD. These ndings provided key information and guidance for further investigation on the pharmacologically active substances and clinical applications of QFPDD. parts of them absorbed then their bioactivites, as and the in the the and the quality and clinical applications of anti-COVID-19 Chinese medicine. This study aims to investigate the main chemical constituents in QFPDD and the absorbed components (including prototypes and metabolites) in serum and tissues after oral administration of QFPDD to mice. To this end, an UHPLC-Q-Orbitrap HRMS based method was well-established for systematic proling the constituents in QFPDD, while the absorbed components (including the prototypes and the metabolites) occurring in mouse serum and tissues were also identied via proling the serum and tissue samples from QFPDD-treated mice. The prototype components in both QFPDD and mouse serum were tentatively identied via comparison with the retention times and MS/MS spectra of the standards or refereed by literature, while the in vivo metabolites were tentatively identied via comparison with the retention times and MS/MS spectra of the in vitro metabolites generated in mice liver microsomes (MLMs). Finally, a total of 405 chemicals (including 40 kinds of alkaloids, 162 kinds of avonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds) in the water extract of QFPDD were identied, while 195, 165, 177, 112, 120, 44, 53 constituents were identied in the serum, lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. All these ndings are very helpful for deep understanding the fates of the constituents in QFPDD, as well as the key information of the tissue distribution of the absorbed components of this anti-COVID-19 Chinese medicine. (p102) in QFPDD, as well as the absorbed prototypes and the metabolites in mice serum and tissues following oral administration of QFPDD. With the help of the authentic standards and in vitro metabolites generated by MLM, a total of 405 constituents (including 40 kinds of alkaloids, 162 kinds of avonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds) were identied from the water extract of QFPDD, while 195 components (including 104 prototypes and 91 metabolites) were identied from mice serum after oral administration of QFPDD to mice. Meanwhile, 165, 177, 112, 120, 44, 53 components were identied from the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. Additionally, the metabolic pathways of some major absorbed components occurring in the serum of QFPDD-treated mice were also described. All these ndings provided key information and guidance for further investigations on the pharmacologically active substances and quality control of QFPDD. quadrupole

Under the optimal conditions described above, the constituents in the crude extract of QFPDD were comprehensively analyzed by UHPLC-Q-Exactive Orbitrap HRMS, with a running time of 45 min. The total ion chromatograms (TICs) of the extract of QFPDD under both positive and negative ion modes are shown in Fig. 1. The protonated molecular weights of all identi ed compounds were calculated within an error of 10 ppm. Following carefully comparison with the retention times and MS/MS spectra of the reference standards, reference literature, Chemical Book and self-bulit databases, a total of 405 chemicals were identi ed or tentatively characterized from QFPDD, including 40 kinds of alkaloids, 162 kinds of avonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds ( Table 1). The detailed information of the identi ed chemical constituents in QFPDD including retention time, precise molecular weight, MS/MS fragment ions and the source of each constituent, have been listed in Table 1. Among all identi ed chemical constituents, ninety-one constituents are con rmed by the authentic standards.  It is well-accepted that deep understanding of the fragmentation pattern of each authentic standard or its analgous is very helpful for the identi cation of herbal constituents by using HRMS [17]. In this study, the fragmentation pathways of various constituents (ephedrine, amygdalin, nobiletin, liquiritin, gallic acid, chlorogenic acid, saikosaponin A and glycyrrhizic acid) in QFPDD were carefully studied and proposed in Fig. 2 and Fig. 3.  Fig. 2A) (Fig. 2C). The compound 321 listed in Table 1   3.2 Characterization of the absorbed chemical constituents in mice serum 3.2.1 Characterization of the absorbed prototype constituents in mice serum Next, the serum samples collected from QFPDD-treated mice at different sampling times (1 h, 2 h and 4 h) were analyzed by UHPLC-Q-Exactive-Orbitrap HRMS to observe the peak numbers and their response in TIC chromatograms. It was found that more peaks could be detected from 1 h serum samples, suggesting that most absorbed prototype constituents or metabolite could be detected in this sample. Thus, the serum samples collected from QFPDD-treated mice at 1 h were collected and mixed for LC-MS analysis to detect the absorbed prototypes and metabolites as more as possible. With the help of the key information of the constituents in QFPDD (as listed in Table 1), a total of 104 prototype compounds were presumed, including 12 kinds of alkaloids, 55 kinds of avonoids, 21 kinds of organic acids, 8 kinds of triterpene saponins and 8 kinds of other compounds (  p-is expressed as prototype; *-is expressed as prototype and con rmed by reference substance; RT-retention time Flavonoids were major constituents in several herbs for preparing QFPDD, it is necessary to identify the absorbed avonoids in serum from QFPDD-treated mice. In this study, a total of 54 avonoids (as the absorbed prototype constituents) were detected in mice serum. Take compound p87 for example, this peak Triterpene saponins were another major group of bioactive components in QFPDD, and it was found that the majority of them could be absorbed into serum.
For example, compound p103 gave molecular formula C 30

Identi cation of the metabolites in mice serum
Next, the metabolites in serum from QFPDD-treated mice that are derived from the constituents in QFPDD were tentatively characterized. To discriminate the endogenous metabolites from QFPDD-related metabolites, the TIC chromatograms of QFPDD-treated mice serum were compared with that of the blank serum.
After then, the endogenous substances could be excluded, while the exogenous compounds could be identi ed by comprehensive analysis. By contrasting these compounds with already known compounds in vitro, searching for the same compounds and validating them via comparison of the retention time and the product ions. TraceFinder software incorporated in UHPLC-Q-Exactive-Orbitrap HRMS could process the data acquired, provide key information about group changes from prototypes to metabolites by means of exact molecular weight, and provide elementary compositions thereof. These analyses were bene cial to determine the prototypes and the metabolites in biological samples, and to conjecture potential metabolic pathways. Furthermore, in this study, In vitro metabolism demonstrated that following incubation of chlorogenic acid in MLMs in the presence of NADPH and SAM, two O-methylated metabolites were detected. The retention times, product ions were well-matched with that of the metabolites M21 and M25. Thus, these two metabolites were identi ed as O-methylated chlorogenic acid. Previous study have reported that two O-methylated metabolites of chlorogenic acid were found in urine samples after intramuscular administration of chlorogenic acid injection to healthy adults, and the pure O-methylated metabolites were synthesized by chlorogenic acid and followed by isolation, puri cation and identi cation [22]. According to the chromatographic behavior of them, M21 was tentatively identi ed as 3′-methyl-chlorogenic acid, and M25 was tentatively identi ed as 4′-methyl-chlorogenic acid.
Following carefully metabolite identi cation as mentioned above, a total of 91 metabolites were tentatively identi ed (Table 3). A variety of metabolic reactions, such as hydroxylation, O-methylation, O-glucuronidation, O-sulfation, demethylation, were involved in the metabolism of the major constituents in QFPDD. Among them, 20 metabolites are fully con rmed by the in vitro metabolites generated in RLM. M-means metabolite, #-means the metabolite that has been con rmed by microsomal incubation in vitro; RT-retention time.

Characterization of the absorbed prototypes and metabolites in mice tissues
With the help of the information about the absorbed prototypes and metabolites in mice serum, the absorbed prototypes and metabolites in various tissues from QFPDD-treated mice were explored. Subsequently, total of 165, 177, 112, 120, 44 and 53 prototypes and metabolites were identi ed in the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. The peak areas of absorbed prototypes and metabolites were summarized in Table 2 and   Table 3, while Fig. 6 showed the heat map of the peak areas of both the absorbed components and the metabolites occurring in serum or six different tissues of QFPDD-treated mice.

Discussion
To ght against COVID-19, some Chinese medicines were recommended by National Health Commission of the People's Republic of China or the local government in China, for alleviating the major symptoms of COVID-19 or preventing disease deterioration [9]. As the rst Chinese medicine compound formula recommended for combating COVID-19, QFPDD is made by 20 herbs and a mineral drug, which is composed by hundreds of ingredients. In most cases, for treating COVID-19, the absorbed constituents (including the prototypes and the metabolites) into blood circulation can be delivered to the target organ (such as the lung) and then exerts various pharmacological effects, such as anti-in ammatory and anticoagulant effects [23,24]. Therefore, it is necessary to investigate the absorbed constituents of QFPDD following oral administration of this super Chinese medicine compound formula, which will be very helpful for better understanding the key material basis of QFPDD, as well as for guiding the quality control and clinical applications of this formula.
In this study, an UHPLC-Q-Orbitrap HRMS based method was established for pro ling the constituents in QFPDD and the absorbed constituents in QFPDDtreated mice. The research strategy and the key ndings for clarifying the chemical composition and absorption components of QFPDD have been summarized in Fig. 7. The chemical constituents in QFPDD were tentatively identi ed via comparison with exact molecular weight, the retention times and MS/MS spectra of the standards or refereed by TCM databases and literature. The absorbed components (including the prototypes and the metabolites) occurring in mice serum and tissues were identi ed via pro ling the serum and tissue samples from QFPDD-treated mice. The absorbed prototype components were tentatively identi ed via comparison with the retention times and MS/MS spectra of components in QFPDD. The in vivo metabolites were tentatively identi ed via comparison with fragment ions of the parent compounds or refereed by literature, while parts of in vivo metabolites of the major constituents were identi ed via comparison with the retention times and MS/MS spectra of the in vitro metabolites of each constituent generated in MLMs. Finally, a total of 405 chemicals (including alkaloids, avonoids, organic acids, triterpene saponins and other compounds) in the extract of QFPDD were identi ed, while 195, 165, 177, 112, 120, 44, 53 constituents were identi ed in the serum, lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. All these ndings might be very helpful for deep understanding the fates of the constituents in QFPDD, as well as offered key information of the tissue distribution of the absorbed components of this anti-COVID-19 Chinese medicine.
It is well known that it is always a challenging task for identi cation of the prototypes and the metabolites of TCM prescription. For example, oroxylin A 7-Oglucuronide (p75) is a trace amount prototype constituent occurring in QFPDD, but this agent is a highly exposed component in the serum and various tissues of QFPDD-treated mice. The reasons may include i) produced by the O-glucuronidation of oroxylin A (p103) in vivo; ii) produced by baicalein (p66) through 6-O-methylation and O-glucuronidation. It is also suggested that it is very di cult to differentiate some components detected in QFPDD-treated mice as the prototypes or the metabolites, owing to that some in vivo components are derived from both the prototypes and the metabolites of the homologous compounds occurring in this TCM prescription. Meanwhile, the exposure of some prototype constituents in blood are extremely low and thus are di cult to be detected, since these natural constituents could be readily transformed into the corresponding metabolites (glucuronides or sulfates) in vivo. For instances, trihydroxy-methoxy avone-O-glucuronides (M35 and M60) and daidzein-sulfates (M33 and M41) can be easily detected in mice serum, but the related prototypes are hardly detected in mice serum. In addition, many nature products have isomers that cannot be distinguished by MS spectral analysis alone, thus it is hard to correlate the parent compounds with the metabolites, such as vanillic acid and its isomer.
Modern pharmacological studies have found that some of absorbed components of QFPDD displayed a wide range of beni cical effects or biological activities. Some pharmacological activities of them are considered to be bene cial for COVID-19 patients. For example, ephedrine, pseudoephedrine and amygdalin displayed signi cant anti-cough and anti-asthmatic effects [25], as well as anti-in ammatory [26,27] and immunomodulatory effects [28,29], which are considered as the major active components of TCM prescriptions for treating respiratory diseases. Zhang et al. [30] report that amygdalin can induce LPS by inhibiting NF-κB and NLRP3 signaling pathways, while baicalin has antiviral, anti-in ammatory, antioxidant, and immunomodulatory pharmacological activities [31]. A recent study has reported that baicalin can signi cantly inhibit the catalytic activity of SARS-CoV-2 3CL pro and then exert antiviral activity [32]. The virtual screening of active ingredients of SARS-CoV-2 virus also suggests that hesperidin and baicalin have potential inhibitory effects [33]. The glycyrrhizin and glycyrrhizic acid in licorice have been found with extensive anti-in ammatory [34] and anti-damage effects [35]. Some previous studies have shown that glycyrrhizin can serve as an alternative agent for treating COVID-19 infection and its associated respiratory syndrome [36][37][38]. The extract of Rhizoma Alismatis processed exerts anti-in ammatory activity via inhibiting cytochrome P450 enzymes [39], the key enzymes responsible for the metabolism of arachidonic acid, thereby reducing the production of in ammatory factors [40][41][42].
Furthermore, some in vivo metabolites of QFPDD are more likely to be the active ingredients. Previous studies clearly demonstrated that a panel of Oglucuronides of avonoids displayed good anticoagulant activities, and their anticoagulant activities are better than that of the prototypes [43]. Recent studies have shown that the blood of COVID-19 patients was in a hypercoagulable state, thus the O-glucuronides of avonoids were more likely to promote blood circulation through anticoagulation and then relieve the symptoms of patients [44][45][46]. This study was not only found a considerable number of prototypes and metabolites in serum, but also found a large number of chemical components in lung, liver and other tissues. Among them, the active ingredients derived from Herba Ephedrae (ephedrine, pseudoephedrine, methylephedrine, phenylpropanolamine etc.), Fructus Aurantii Immaturus (stachydrine, naringin, hesperidin, neohesperidin, quinic acid etc.), Radix Scutellariae (baicalin, oroxylin A 7-O-glucuronide, wogonoside, wogonin, oroxylin A etc.) and Rhizoma Alismatis (alisol O, alisol A etc.) displayed relatively high exposure levels in most of organs. However, the kinds and exposure levels of each component in various tissues are much different. These ndings suggest that these in vivo metabolites of QFPDD may play potential role in the treatment of COVID-19 via targeting multiple targets or pathways. In future, the bioactive ingredients and mechanisms of action can be explored.

Conclusion
In summary, a practical and sensitive UHPLC-Q-Exactive-Orbitrap HRMS approach was developed for chemical pro ling of the constituents in QFPDD, as well as the absorbed prototypes and the metabolites in mice serum and tissues following oral administration of QFPDD. With the help of the authentic standards and in vitro metabolites generated by MLM, a total of 405 constituents (including 40 kinds of alkaloids, 162 kinds of avonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds) were identi ed from the water extract of QFPDD, while 195 components (including 104 prototypes and 91 metabolites) were identi ed from mice serum after oral administration of QFPDD to mice. Meanwhile, 165, 177, 112, 120, 44, 53 components were identi ed from the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. Additionally, the metabolic pathways of some major absorbed components occurring in the serum of QFPDD-treated mice were also described. All these ndings provided key information and guidance for further investigations on the pharmacologically active substances and quality control of QFPDD.