Abstract Licorice is well known for its ability to reduce the toxicity of the whole prescription in traditional Chinese medicine theory. However, honey-fired licorice (ZGC for short), which is made of licorice after being stir-fried with honey water, is more commonly used for clinical practice. The metabolism in vivo and detoxification-related compounds of ZGC have not been fully elucidated. In this work, the chemical constituents in ZGC and its metabolic profile in rats were both identified by high ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The network pharmacology was applied to predict the potential detoxifying ingredients of ZGC. As a result, a total of 115 chemical compounds were identified or tentatively characterized in ZGC aqueous extract, and 232 xenobiotics (70 prototypes and 162 metabolites) were identified in serum, heart, liver, kidneys, feces, and urine. Furthermore, 41 compounds absorbed in serum, heart, liver, and kidneys were employed for exploring the detoxification of ZGC by network pharmacology. Ultimately, 13 compounds (five prototypes including P5, P24, P30, P41 and P44, and 8 phase Ⅰ metabolites including M23, M47, M53, M93, M100, M106, M118, and M134) and nine targets were anticipated to be potential mediums regulating detoxification actions. The network pharmacology analysis had shown that the ZGC could detoxify mainly through regulating the related targets of cytochrome P450 and glutathione. In summary, this study would help reveal potential active ingredients in vivo for detoxification of ZGC and provided practical evidence for explaining the theory of traditional Chinese medicine with modern technology. Keywords: honey-fired licorice, UPLC-Q-TOF-MS, metabolism, network pharmacology, detoxification 1 Introduction Glycyrrhizae Radix et Rhizoma, also named Gancao in Chinese or licorice in English, is the dried root and rhizoma of Glycyrrhiza uralensis Fisch. or G. glabra L. or G. inflata Bat. from the family Leguminosae ([39]Li et al., 2019). Licorice is a traditional herb medicine that is regarded as “the progenitor of herbs” ([40]Maria Pia et al., 2018) and has a unique function of detoxification. After stir-fried licorice with honey water, honey-fired licorice (short for ZGC) is obtained. It was originally mentioned in a traditional Chinese medicine (TCM) classics named “Treatise on Cold Damage” written by Zhongjing Zhang in the 3rd century. The chemical composition of licorice changed after processing with honey. Honey used as an adjuvant can accelerate the mutual transformation of liquiritin and isoliquirtin in ZGC ([41]Ota and Makino, 2022). In addition, the content of liquiritin, liquiritin apioside, and glycyrrhizin in ZGC has significantly reduced as well ([42]Wang et al., 2012). Concurrently, ZGC is widely used for detoxification in clinic based on TCM theory. In TCM theory, the detoxification of licorice could be interpreted as the synergistic effect of reducing the toxicity of toxic herbs and the increased tolerance of toxicity in prescription. Licorice has been widely observed in combined usage with other herbal medicines, including Aconiti Lateralis Radix Praeparata ([43]Sun et al., 2016), Aconiti Kusnezoffii Radix ([44]Yan et al., 2017), Sophorae Flavescentis Radix ([45]Shi et al., 2015), Strychni Semen ([46]Gu et al., 2014), and so on. Moreover, it may contribute to reducing oxidative stress and further alleviating toxicity through regulation of the expression cytochrome P450 (CYP450) ([47]Liu et al., 2019). The published study indicated that the flavonoids and pentacyclic triterpene saponin were the major constituents of the licorice combined with toxic substances for the detoxification of licorice ([48]Liu et al., 2019). Liquiritigenin and isoliquiritigenin generally exhibited potent inhibitory bioactivities against the CYP1A2 and CYP2C9 ([49]Li et al., 2019). Glycyrrhetic acid-related metabolites could be induced by CYP3A4 ([50]Hou et al., 2012). In addition, glycyrrhizin and glycyrrhetinic acid have a powerful antioxidant effect which could scavenge free radicals and further reduce the cardiac-toxic lipid peroxidation reaction ([51]Chan et al., 2021). Nevertheless, the detoxification ingredients of ZGC have not been fully elucidated. The research on the metabolic profile of drugs is valuable for a better understanding of the process which contains absorption, distribution, metabolism, and excretion after administration. The published studies about the metabolism of licorice have almost focused on serum, urine, and feces ([52]Huang et al., 2018). Since heart, liver, and kidneys are vital organs for acting detoxification. Thus, it is of crucial interest to establish a sensitive and effective analytical method for a systematic study on the metabolic profile of ZGC in rat serum, heart, liver, kidneys, urine, and feces. In the present study, a fast and selective ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was applied to characterize the chemical compounds of ZGC aqueous extract and metabolic profile in vivo (serum, heart, liver, kidneys, feces, and urine). Next, the prototypes and phase Ⅰ metabolites identified in serum, heart, liver and kidneys were screened for network pharmacology to explore the potential ingredients of detoxification in ZGC. The workflow was shown in [53]Figure 1. FIGURE 1. [54]FIGURE 1 [55]Open in a new tab Workflow for metabolic profile and potential ingredients detoxification of ZGC. 2 Materials and Methods 2.1 Materials and Reagents ZGC (batch number: 181201191) was obtained from Kangmei Pharmaceutical Limited Corporation Co., Ltd (Nei Mongo, China). All ZGC samples were authenticated by associated professor Haibo Huang (Guangzhou University of Chinese Medicine, Guangzhou, China). The reference standards for liquiritin (batch number: RP200207), liquiritin apioside (batch number: RP200107), ononin (batch number: RP200209), isoliquiritin (batch number: RP190308), liquiritigenin (batch number: RP200702), isoliquiritigenin (batch number: RP210115), glycyrrhizic acid (batch number: RP190517), glycyrrhizic acid ammonium salt (batch number: RP201104), and glycyrrhetinic acid (batch number: RP190512) were obtained from Chengdu Madsen Technology Co., Ltd. Chromatographic grade methanol (batch number: I1077207008), chmatographic acetonitrile (lot number: [56]JB079830) and chromatformic acid (batch number: H158A) were purchased from Honeywell Trading Co., Ltd (Shanghai, China). Purified water was supplied by Watsons (Hong Kong, China). Oasis HLB solid-phase extraction (SPE) cartridge (6 cc/200 mg, 30 μm) was brought from Waters Technologies Co., Ltd. (Milford, America). 2.2 Standard Solutions All the individual reference standard stock solutions in methanol were prepared with appropriate concentration, Then, the stock standard solutions were further diluted appropriately by methanol to prepare the mixed standard solution. The concentrations of liquiritin, liquiritin apioside, ononin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhizic acid ammonium salt, and glycyrrhetinic acid were 54.2, 99.8, 102.1, 87.3, 89.0, 56.3, 106.3, 79.3, and 102.3 μg/ml, respectively. 2.3 Preparation of ZGC Aqueous Extract ZGC (30 g) was weighed and soaked in 300 ml water for 30 min at room temperature. After boiling for 2 h, the extract solution was filtered and concentrated to 50 ml. Finally, the ZGC aqueous extract at a concentration of 0.6 g/ml was prepared. 2.4 Animals and Drug Administration Twelve healthy male rats weighing 220–250 g were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). This study was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (No. 20190926003). The animals were bred in the Class SPF Experimental Animal Room of Guangzhou University of Chinese Medicine for a week. All of the rats were kept in an environment at a temperature of 20 ± 2°C with the dark-light for 12 h. Food and water were provided spontaneously to rats for 2 weeks. Before the experiment, these animals were fasted with free access to water in cages separately overnight. Twelve rats were randomized into two groups: blank and ZGC-dosed group. ZGC aqueous extract was given to rats at a dosage of 7.2 g/kg twice a day ([57]Tan et al., 2010; [58]Xiang et al., 2011; [59]Qiao et al., 2012) for three continuous days. At the same time, the blank group was given the same dosage of normal saline. Three rats in each group were used for sampling serum, heart, liver, kidneys, and the other three rats were used for sampling urine and feces. 2.5 Biological Samples Collection and Pretreatment 2.5.1 Serum Samples The blood samples of rats (n = 3) ([60]Lin et al., 2018; [61]Hu et al., 2019) were collected from the orbital venous plexus into tubes at 0.25, 0.5, 1, 2, 4h after the last administration, then separated by centrifuging at 4,000 rpm for 10 min to attain serum. The pooled serum sample (2 ml) was processed 4-fold volume of methanol to precipitate the protein, followed by vortexing for 2 min and centrifuging at 14,000 rpm for 10 min to obtain the supernatant. The supernatant was dried through vacuum centrifugal concentrator on AL mode vent at 30°C, the supernatant was dried. The residues were reconstituted with 200 μl of 50% acetonitrile, vortexed for 1 min, and centrifuged at 14,000 rpm for 10 min. Finally, a 2 μl supernatant was injected into UPLC-Q-TOF-MS for analysis. 2.5.2 Samples of Heart, Liver and Kidneys Each group of rats (n = 3) was sacrificed at the last time point. The heart, liver, and kidneys were removed and washed with normal saline. The tissue samples (0.1 g) were homogenized in 1 ml normal saline under the condition of ice bath. The mixture was then centrifuged at 12,000 rpm for 10 min at 4°C to obtain the supernatant. Then, the 4-fold volume of methanol was added to the supernatant solution obtained to precipitate protein, followed by vortexing for 2 min and centrifuging at 12,000 rpm for 10 min. Through vacuum centrifugal concentrator on AL mode vent at 30°C, the supernatant was dried. The residues were reconstituted with 200 μl of 50% acetonitrile and vortexed for 1 min and centrifuged at 12,000 rpm for 10 min. Subsequently, 1 μl supernatant was injected into the UPLC-Q-TOF-MS system for analysis. 2.5.3 Urine Samples The urine samples of rats (n = 3) were collected from 0 to 12 h and 12–24 h after administration. The pooled urine sample (6 ml) was centrifuged at 4,000 rpm for 10 min. Then the 6 ml supernatant urine samples were added to pre-treated SPE columns and eluted with 0.1% formic acid and methanol, and washed with pure water once which would be discarded then. Then 0.1% formic acid and methanol were applied as the eluent for elution, with the further elution solution being collected. Through vacuum centrifugal concentrator on AL mode vent at 30°C, the supernatant was dried. The residues were reconstituted with 200 μl of 50% acetonitrile, vortexed for 1 min, and centrifuged at 14,000 rpm for 10 min. Finally, a 2 μl supernatant was injected into UPLC-Q-TOF-MS for analysis. 2.5.4 Feces Samples The feces samples of rats (n = 3) were collected from 0 to 12 h and 12–24 h after administration. All feces samples of the two periods were mixed and dried. The feces samples (1 g) were extracted with methanol, on which the ultrasonic was performed for 30 min under the condition of 500 W and 40 kHz, before being centrifuged at 14,000 rpm for 10 min. Through vacuum centrifugal concentrator on AL mode vent at 30°C, the supernatant was dried. The residues were reconstituted with 50% acetonitrile, vortexed for 1 min, and centrifuged at 14,000 rpm for 10 min. Finally, a 2 μl supernatant was injected into UPLC-Q-TOF-MS for analysis. 2.6 Instrument and Analysis Conditions All samples were analyzed on an AB Sciex 5600 Triple-TOF™ mass spectrometer (AB Sciex, CA, United States) coupled with a Shimadzu UPLC LC-30AD system (Kyoto, Japan) and controlled with the Analyst TF 1.7 software (AB Sciex). Chromatographic separation was carried out at 30°C using a Hypersil GOLD aQ C18 column (Waters, United States; 2.1 mm × 100 mm, 1.9 μm). The mobile phase consisted of acetonitrile (A) and water (B) (both including 0.1% aqueous formic acid, v/v), and the flow rate was 0.4 ml/min. The gradient elution was performed as follows: 95% B at 0.0–1.0 min, 95–90% B at 1.0–2.0 min, 90–85% B at 2.0–4.0 min, 85–80% B at 4.0–6.0 min, 80–75% B at 6.0–8.0 min,75–65%B at 8.0–15.0 min, 65–55% B at 15.0–20.0 min, 55–5% B at 20.0–24.0 min, 5–0% B at 24.0–25.0 min, 0% B at 25.0–26.0 min. The mass spectrometer was coupled with an electrospray ionization (ESI) source, acquiring TOF-MS and TOF-MS-IDA-MS/MS spectra in the dynamic background subtraction by running in negative ion and high sensitivity mode. In addition, the automated calibration delivery system could acquire precise mass measurements. The operating parameters were as follows: Behind optimization, nitrogen was used as a source of gas, and the nebulizer gas, curtain gas, and heater gas were set at 55, 35 and 55 psi, respectively. The source temperature, ion spray voltage (ESI^+/ESI^−), and declustering potential were set at 550°C, 5500/−4500 V and 100/−100 V, respectively. For the IDA experiments, the collision energies were set at −45 and 45 eV, and the collision energy spread was set at 10/15 eV. TOF-MS spectra were obtained from 100 to 2,000 Da. 2.7 UPLC-Q-TOF-MS Data Processing The self-building chemical library including compound name, molecular formula, structure and accurate molecular mass was established by literature, PubMed, Chemspider, CNKI, SciFinder, in which every single compound was numbered. The characteristic chromatographic behaviors of available reference standards were obtained to identify the unknown compounds in ZGC aqueous extract. The molecular formula and order number of chemicals from the self-built library were imported into Peakview 1.5 to identify the chemical constituents of ZGC. The most rational molecular formula and the mass accuracy within a reasonable degree of measurement error (ppm <5) were gained. Meanwhile, compounds were identified by the fragmentation information obtained. Finally, LC-MS data file in positive and negative ion modes was applied to characterize ZGC-related xenobiotics (prototypes and metabolites) for biosamples from rats being administrated with ZGC. The representative prototypes were imported into MetabolitePilot 2.0.4 to search and identify the metabolites along with their metabolic pathways. The useful clogP values of compounds calculated by ChemDraw Ultra 19.0 was introduced to distinguish between structural isomers. In general, compounds had a longer retention time when the values of clogP were higher in reversed-phase liquid chromatography systems ([62]Hallgas et al., 2004; [63]Liang et al., 2013). 2.8 Network Pharmacology Study 2.8.1 Prediction of Compounds and Targets of Detoxification in ZGC The compounds (prototypes and phase Ⅰ metabolites) of ZGC identified from serum, heart, liver, and kidneys were imported into PharmMapper (Z′-score ≥ 1.5, [64]http://www.lilab-ecust.cn/pharmmapper/) ([65]Zhou et al., 2019) and Swiss Target Prediction (top 15, [66]http://www.swisstargetprediction.ch/) ([67]Daina et al., 2019) to predict the targets, respectively. Then, the corresponding target and uniport ID were obtained through Uniprot database ([68]http://www.uniprot.org/). “Detoxify” were imported into GeneCards database ([69]https://www.genecards.org/) to collect the detoxification-related targets. The potential detoxification targets of ZGC were selected using Protein–Protein Interaction (PPI) with a high-combining score of more than 0.900 ([70]Huang et al., 2019). The key compounds and targets were screened with the medians of Betweenness Centrality (BC), Closeness Centrality (CC), and Degree Centrality (DC) respectively by NetworkAnalyzer (a plug-in of Cytoscape). 2.8.2 GO and KEGG Pathway Enrichment Based on the mechanism of detoxification, the detoxification-related targets were further screened by existing literature. OmicShare ([71]https://www.omicshare.com/) was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The cut-off criterion of those GO and pathway terms was p-value < 0.05 ([72]Qi et al., 2019), which was considered statistically significant. 3 Results 3.1 Determination of Ingredients in ZGC by HPLC In this study, according to the method of the Chinese Pharmacopoeia 2020 edition, the ingredients in ZGC assayed by HPLC, the two content-determined compounds of ZGC, were liquiritin (6.11 mg/ml) and glycyrrhizic acid (33.88 mg/ml). 3.2 Identification and Characterization of Chemical Compounds The exposed chemical components were identified by referring to relative retention time, accurate m/z values of quasimolecular ion, characteristic fragment ion, mass fragmentation rules from available databases and by comparing mass data with those from authentic standards and previous studies ([73]Infante et al., 2012; [74]He et al., 2014; [75]Qiao et al., 2015). A total of six flavonoids and three pentacyclic triterpenoid saponins were exactly identified with reference standards, including liquiritin, liquiritin apioside, ononin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhizic acid ammonium salt, and glyrrhetinic acid. The chemical structures of nine reference standards of ZGC were shown in [76]Supplementary Figure S1, and the detailed MS information of nine reference standards in ZGC was shown in [77]Supplementary Table S1. The extracted ion chromatograms (EICs) of nine reference standards of ZGC were shown in [78]Supplementary Figure S2. The fragment pathways of these chemical compounds have been well elucidated in published references ([79]Xu et al., 2013; [80]Zhou et al., 2013; [81]Zhang et