key: cord-0837595-899ai4my
authors: Lan, Xiao-fang; Olaleye, Olajide E.; Lu, Jun-lan; Yang, Wei; Du, Fei-fei; Yang, Jun-ling; Cheng, Chen; Shi, Yan-hong; Wang, Feng-qing; Zeng, Xue-shan; Tian, Nan-nan; Liao, Pei-wei; Yu, Xuan; Xu, Fang; Li, Ying-fei; Wang, Hong-tao; Zhang, Nai-xia; Jia, Wei-wei; Li, Chuan
title: Pharmacokinetics-based identification of pseudoaldosterogenic compounds originating from Glycyrrhiza uralensis roots (Gancao) after dosing LianhuaQingwen capsule
date: 2021-04-30
journal: Acta Pharmacol Sin
DOI: 10.1038/s41401-021-00651-2
sha: a53134e5b3350b3f94a1a754320ff04f66933c8d
doc_id: 837595
cord_uid: 899ai4my

LianhuaQingwen capsule, prepared from an herbal combination, is officially recommended as treatment for COVID-19 in China. Of the serial pharmacokinetic investigations we designed to facilitate identifying LianhuaQingwen compounds that are likely to be therapeutically important, the current investigation focused on the component Glycyrrhiza uralensis roots (Gancao). Besides its function in COVID-19 treatment, Gancao is able to induce pseudoaldosteronism by inhibiting renal 11β-HSD2. Systemic and colon-luminal exposure to Gancao compounds were characterized in volunteers receiving LianhuaQingwen and by in vitro metabolism studies. Access of Gancao compounds to 11β-HSD2 was characterized using human/rat, in vitro transport, and plasma protein binding studies, while 11β-HSD2 inhibition was assessed using human kidney microsomes. LianhuaQingwen contained a total of 41 Gancao constituents (0.01–8.56 μmol/day). Although glycyrrhizin (1), licorice saponin G2 (2), and liquiritin/liquiritin apioside (21/22) were the major Gancao constituents in LianhuaQingwen, their poor intestinal absorption and access to colonic microbiota resulted in significant levels of their respective deglycosylated metabolites glycyrrhetic acid (8), 24-hydroxyglycyrrhetic acid (M2(D); a new Gancao metabolite), and liquiritigenin (27) in human plasma and feces after dosing. These circulating metabolites were glucuronized/sulfated in the liver and then excreted into bile. Hepatic oxidation of 8 also yielded M2(D). Circulating 8 and M2(D), having good membrane permeability, could access (via passive tubular reabsorption) and inhibit renal 11β-HSD2. Collectively, 1 and 2 were metabolically activated to the pseudoaldosterogenic compounds 8 and M2(D). This investigation, together with such investigations of other components, has implications for precisely defining therapeutic benefit of LianhuaQingwen and conditions for its safe use.

Chinese traditional medicine has played an important role in management of the coronavirus disease 2019 (COVID-19) [1] . LianhuaQingwen capsule is a Chinese herbal medicine recommended in the official guideline for the diagnosis and treatment of COVID-19 [2] . The capsule is prepared from a combination, comprising Forsythia suspensa fruits (Lianqiao), Lonicera japonica flower buds (Jinyinhua), Ephedra sinica stems stir-fried with honey (Zhi-Mahuang), stir-fried Prunus sibirica seeds (Chao-Kuxinren), Gypsum Fibrosum (Shigao), Isatis indigotica roots (Banlangen), Dryopteris crassirhizoma rhizome (Mianmaguanzhong), Houttuynia cordata whole plants (Yuxingcao), Pogostemon cablin overground portion (Guanghuoxiang), Rheum palmatum rhizomes and roots (Dahuang), Rhodiola crenulata rhizomes and roots (Hongjingtian), l-menthol (Bohenao), and Glycyrrhiza uralensis roots (Gancao). In a recent prospective, multicenter, randomized controlled trial of 284 patients with COVID-19, adding LianhuaQingwen to conventional care further improved the recovery rate of symptoms, shortened the time to symptom recovery, and improved the recovery of chest radiologic abnormalities (P < 0.05 for all) [3] . Two retrospective observational studies showed that adding the capsule to conventional care further accelerated alleviation of the symptoms fever, cough, and fatigue (P < 0.05) [4, 5] . In a cell-based study, LianhuaQingwen dose-dependently reduced production of proinflammatory cytokines at mRNA level, inhibited the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, and induced abnormal particle morphology of virion [6] . In addition, several circulating constituents (including rhein, forsythoside A, forsythoside I, and neochlorogenic acid) of LianhuaQingwen were identified to block the host target cell-surface protein angiotensinconverting enzyme 2 receptor [7] , to which the SARS-CoV-2 spike protein binds. Like for COVID-19, LianhuaQingwen was also used, in China, as treatment for other acute viral respiratory illnesses, including 2003 severe acute respiratory syndrome (SARS), 2009 H1N1 swine influenza, 2013 Middle East respiratory syndrome, and 2013 H7N9 avian influenza [8, 9] .

Despite extensive use as treatment for acute viral respiratory illnesses, LianhuaQingwen needs to be subjected to more objective rigorous evaluation procedures to provide scientific evidence for its use. An understanding of chemical composition and pharmacokinetics and disposition of key constituents (bioavailable at loci responsible for the therapeutic and/or adverse effects) is vital for translating the potential benefits of complex LianhuaQingwen in rigorous clinical trials. Over the past decade, we have seen great advances in methodology and associated techniques for pharmacokinetic research on such Chinese herbal medicines [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . The field now provides methods that are ripe for use to facilitate identifying the key constituents responsible for therapeutic effects, adverse effects, or drug-drug interactions of such medicines [22] [23] [24] [25] . For a systemically acting drug for acute respiratory viral infection, systemic exposure is a prerequisite for drug molecules' access to the loci responsible for the therapeutic effects (such as antiviral effects). Intestinal microbiota plays a vital role in human metabolism, immunity, and reactions to diseases [26] . Acute respiratory viral infection can induce intestinal microbiome alterations and secondary bacterial pneumonia, and for the treatment of patients with COVID-19 is recommended in China to preserve intestinal balance and to prevent secondary bacterial infections [27] [28] [29] . Thus, pharmacokinetic research on LianhuaQingwen entails (i) search for herbal compounds circulating significantly (i.e., systemic exposure) after doing the capsule and evaluation of their pharmacokinetics and disposition (including lung distribution) and (ii) evaluation of access of the capsule's compounds to colonic microbiota (i.e., colon-luminal exposure).

As a part of our serial pharmacokinetic investigations of LianhuaQingwen, the current investigation focused on constituents, originating from the component Gancao (licorice), after dosing the capsule. Glycyrrhizin is a known bioactive constituent of Gancao and was found to be active in inhibiting replication of the SARS-associated virus [30] . Notably, pseudoaldosteronism, characterized by hypokalaemia, hypertension, and peripheral edema, could be an adverse effect of consuming Gancaocontaining preparations, by inhibiting renal 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) [31] . Several glycyrrhizin metabolites, i.e., glycyrrhetic acid-3-O-glucuronide [32] [33] [34] [35] [36] , glycyrrhetic acid-3-O-sulfate [37, 38] , 22α-hydroxyglycyrrhetic acid-3-O-sulfate [37] , and 22α-hydroxyglycyrrhetic acid-3-O-sulfate-30-O-glucuronide [39] , have been inconsistently proposed to be responsible for or as biomarkers of Gancao-induced pseudoaldosteronism. Given that Gancao is widely used as a component in Chinese herbal medicines and also in dietary products, more research is needed to clarify the compounds responsible for the Gancao-induced adverse effect; this has implication for precisely defining conditions for safe use of LianhuaQingwen. This pharmacokinetic investigation aimed to identify, with respect to systemic exposure and colon-luminal exposure, bioavailable Gancao compounds that are likely to influence the therapeutic outcomes of LianhuaQingwen, particularly potential pseudoaldosterogenic Gancao compounds. Given that the therapeutic action of LianhuaQingwen is also attributed to other components, identified Gancao compounds were not further evaluated in this investigation with respect to their bioactivities associated with the therapeutic action.

A detailed description of materials and experimental procedures is provided in the Supplementary Materials and Methods.

Study design This investigation of LianhuaQingwen capsule was designed to facilitate identifying Gancao compounds that are likely to be therapeutically important (i.e., bioavailable at loci responsible for drug response of the capsule), particularly pseudoaldosterogenic Gancao compounds. Bioavailable Gancao compounds were identified in human volunteers receiving LianhuaQingwen at label daily dose, with respect to systemic and colon-luminal exposure to the compounds. Because pseudoaldosteronism is a Gancaospecific adverse effect, pseudoaldosterogenic compounds were identified from circulating Gancao compounds of LianhuaQingwen, based on their access to and inhibition of renal 11β-HSD2. Systemic exposure to Gancao compounds was investigated, based on a full understanding of Gancao-related chemical composition of the dosed LianhuaQingwen. Unchanged and metabolized Gancao compounds in the human plasma and urine samples were characterized by liquid chromatography/mass spectrometry. Gancao metabolites were characterized by various in vitro metabolism studies as well. Colon-luminal exposure to Gancao compounds was characterized by analyzing the human fecal samples and also by in vitro study of metabolism of unabsorbed Gancao constituents by colonic microbiota. Access of circulating Gancao compounds to renal 11β-HSD2 was characterized by evaluating their renal clearance ratio, plasma protein binding, membrane permeability, and influences of renal transporters. Inhibition of 11β-HSD2 by the circulating Gancao compounds was assessed using human kidney microsomes. Several rat studies were performed to facilitate a better understanding of factors influencing systemic exposure to Gancao metabolites and the metabolites' pulmonary exposure and access to renal 11β-HSD2; associated interspecies similarities and differences between rats and humans were evaluated before using the rat data. Literature mining was performed before the preceding in vivo and in vitro studies to avoid missing any potentially important Gancao-related compounds. Fig. 1 summarizes the study workflow.

Literature mining Literature mining, for supporting experimental parts of this investigation, was performed to obtain three types of information: (i) various constituents of Glycyrrhiza species and their presence in LianhuaQingwen, (ii) pharmacokinetics, intestinal absorption, and disposition of Gancao compounds and their interactions with colonic microbiota, and (iii) Gancao-induced pseudoaldosteronism. A detailed method for literature mining is described in the Supplementary Materials and Methods.

LianhuaQingwen and study materials LianhuaQingwen capsule was manufactured by Shijiazhuang Yiling Pharmaceutical Co., Ltd (Yiling Pharmaceutical; Shijiazhuang, Hebei Province, China) with a Chinese NMPA drug ratification number of GuoYaoZhunZi-Z20040063. Samples of 29 lots of LianhuaQingwen were obtained from Yiling Pharmaceutical to determine Gancao-related chemical composition and lot-to-lot variability. Methods for the chemical composition analysis and for the lot-to-lot variability evaluation are described in subsection 2.9. Samples of the components, including Gancao (G. uralensis roots), were also obtained from Yiling Pharmaceutical. Pure compounds reported to be present in G. species were obtained commercially (Supplementary Table S1 ). 24-Hydroxyglycyrrhetic acid was prepared via anaerobic deglycosylation of licorice saponin G2 using rat colonic microbiota. Glycyrrhetic acid-30-O-glucuronide was prepared via glucuronidation of glycyrrhetic acid using UDPGA-fortified rat liver microsomes.

Modified Gifu anaerobic medium broth was obtained from HyServe (Uffing, Germany). Pooled human liver microsomes (HLM), pooled human intestinal microsomes (HIM), pooled human liver cytosol (HLC), and pooled human intestinal cytosol (HIC) were obtained from Corning Gentest (Woburn, MA, USA).

Pooled human kidney microsomes (HKM) was obtained from Celsis In Vitro Technologies (Baltimore, MD, USA) and Sekisui XenoTech (Kansas City, KS, USA); the donor information is shown in Supplementary Table S2 . Pooled rat liver microsomes (RLM), pooled rat intestinal microsomes (RIM), pooled rat liver cytosol (RLC), and pooled rat kidney microsomes (RKM) were prepared inhouse from livers, intestines, and kidneys of male Sprague-Dawley rats by differential centrifugation.

HEK-293 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Human solute carrier transporter expression plasmids were constructed commercially. Inside-out membrane vesicles expressing human ATP-binding cassette transporter were purchased from GenoMembrane (Kanazawa, Japan). The positive substrates for these enzymes and transporters were obtained commercially.

Human pharmacokinetic study of LianhuaQingwen A single-center, open-label pharmacokinetic study of Lianhua-Qingwen was performed in healthy volunteers at Hebei Yiling Hospital (Shijiazhuang, Hebei Province, China). The protocol for study was reviewed and approved by an ethics committee of clinical investigation at the hospital. The study was registered at the Chinese Clinical Trials Registry (www.chictr.org.cn) with a registration number of ChiCTR1900021460 and performed in accordance with the Declaration of Helsinki. All volunteers gave written informed consent prior to enrollment.

Each participant orally received a single dose of 12 capsules (label daily dose; taking 12 capsules with 240 mL water within 1 min in three swallows with four capsules each) of LianhuaQingwen on day 1 and days 3-8. On day 1, serial blood samples (5 mL) were collected before and 0.167, 0.5, 1, 3, 6, 9, 12, 16, 24, 28, 34, and 48 h after dosing the 12 capsules, while serial urine samples were also collected within 0-4, 4-10, 10-24, 24-32, and 32-48 h periods after dosing. On days 3-7, blood samplings were performed before and 12 h after dosing the daily 12 capsules. On day 8, blood and urine samplings were performed according to the respective time schedules on day 1. On the day just before day 1 (i.e., day 0) and on day 9 or 10, fecal samples (3-5 g) were freshly collected from the participants and immediately mixed with fivefold volumes of the modified Gifu anaerobic medium followed by centrifuging at 1000 × g for 5 min and the supernatants containing the colonic microbiota were collected under anaerobic conditions to yield individual samples of human colonic microbiota (HCM). The individual samples (1 mL) on day 0 from the participants were combined to yield a pooled preparation of HCM. All the human study samples were analyzed by liquid chromatography/mass spectrometry.

The final concentrations of the test substrates in the in vitro metabolism studies were designed to allow the metabolites to be reliably measured. Multiple final concentrations of the test substrates were used for each enzyme kinetic study.

Deglycosylation by colonic microbiota. Glycyrrhizin (1), licorice saponin G2 (2), liquiritin (21) , liquiritin apioside (22) , isoliquiritin apioside (23) , and isoliquiritin (24) were separately dissolved in the modified Gifu anaerobic medium. Before use, the glycosidase activities of pooled HCM and pooled rat colonic microbiota (RCM) preparations were tested under anaerobic conditions using hesperidin as positive substrate [40] . After anaerobic preincubation at 37°C for 1 h, the HCM preparation (500 μL) was mixed with equal volume of the test compound solution with final compound concentration at 1 μmol/L followed by incubating anaerobically at 37°C for 4 h. Sampling was performed at 10 and 30 min and 4 h after initiating incubation and the samples were mixed with two volumes of ice-cold methanol. After centrifugation, the supernatant sample was analyzed by liquid chromatography/mass spectrometry. The anaerobic incubation was performed in a Labiophy AL-B anaerobic workstation (Dalian, Liaoning Province, China), inflated with a gas mix of~15% carbon dioxide, 83% nitrogen, and 2% hydrogen. This study was repeated but by using the pooled preparation of RCM.

Deglycosylation by hepatic glycosidases. Glycyrrhizin (1) at 10 µmol/L was incubated with HLC at 37°C for 20 h (as described previously [22] ) and the resulting incubation sample was analyzed by liquid chromatography/mass spectrometry. This study was repeated but by using RLC.

Oxidation by intestinal and hepatic P450s. Glycyrrhetic acid (8) at 1 µmol/L was incubated with NADPH-fortified HLM at 37°C for 1 h (as described previously [41] ) and the resulting incubation sample was analyzed by liquid chromatography/mass spectrometry. This study was repeated but by using NADPH-fortified HIM and RLM. Kinetics of hydroxylation of 8 mediated by P450s in HLM and RLM was assessed with respect to Michaelis constant (K m ), maximum velocity (V max ), and intrinsic clearance (CL int ). The assessment was conducted under linear metabolism conditions by incubation for 15 min, and the test concentrations of 8 were 0.78-100 μmol/L for HLM and RLM.

Conjugation by intestinal and hepatic UGTs and SULTs. Glycyrrhetic acid (8), 24-hydroxyglycyrrhetic acid (M2 D ), and liquiritigenin (27) , each at 1 µmol/L, were separately incubated with UDPGA-fortified HLM at 37°C for 1 h (as described previously [19, 25] ) and the resulting incubation samples were analyzed by liquid chromatography/mass spectrometry. This study was repeated but by using UDPGA-fortified HIM, RLM, and RIM. Kinetics of glucuronidation of 8 mediated by UGTs in HLM and RLM was assessed with respect to K m , V max , and CL int . The assessment was conducted under linear metabolism conditions by incubation for 5 min, and the test concentrations of 8 were 1.56-200 and 0.78-100 μmol/L for HLM and RLM, respectively.

Glycyrrhetic acid (8), 24-hydroxyglycyrrhetic acid (M2 D ), glycyrrhetic acid-30-O-glucuronide (M8 G ), and liquiritigenin (27) at 1 µmol/L for each were incubated with PAPS-fortified HLC at 37°C for 1 h (as described previously [42] ) and the resulting incubation samples were analyzed by liquid chromatography/mass spectrometry. This study was repeated but by using PAPS-fortified HIC and RLC. Kinetics of sulfation of 8 mediated by SULTs in HLC and RLC was assessed with respect to K m , V max , and CL int . The assessment was conducted under linear metabolism conditions by incubation for 15 min, and the test concentrations of 8 were 0.27-200 and 2.47-200 μmol/L for HLC and RLC, respectively. Two-step metabolism by human hepatic enzymes. To assess twostep metabolism of 24-hydroxyglycyrrhetic acid (M2 D ) (i.e., conjugation → conjugation), the compound at 10 µmol/L was first incubated with UDPGA-fortified HLM or PAPS-fortified HLC at 37°C for 1 h and then incubated with PAPS-fortified HLC or UDPGA-fortified HLM, respectively, at 37°C for 2 h. The final incubation samples were analyzed by liquid chromatography/ mass spectrometry.

Supportive in vitro transport studies of Gancao compounds Cell cultures and cellular uptake by human OAT1, OAT2, OAT3, OAT4, OCT2, OCTN1, URAT1, PEPT1, and PEPT2 were assessed for glycyrrhetic acid (8), 24-hydroxyglycyrrhetic acid (M2 D ), glycyrrhizin (1), glycyrrhetic acid-3-O-glucuronide (7), glycyrrhetic acid-30-O-glucuronide (M8 G ), liquiritin (21) , liquiritin apioside (22) , and liquiritigenin (27) , each at 50 µmol/L, as described previously [43] . In addition, membrane vesicles expressing human MRP3 or MRP4 were used to assess transport of 1, 7, and M8 G , each at 20 µmol/L, as described previously [44] . All the incubation samples were analyzed by liquid chromatography/mass spectrometry.

Supportive in vitro plasma-protein-binding studies of Gancao compounds Unbound fractions in plasma were assessed for glycyrrhetic acid (8), 24-hydroxyglycyrrhetic acid (M2 D ), glycyrrhizin (1), glycyrrhetic acid-3-O-glucuronide (7), glycyrrhetic acid-30-O-glucuronide (M8 G ), and liquiritigenin (27) using an equilibrium dialysis method, as described previously [45] . In addition, human albumin (600 μmol/L), α 1 -acid glycoprotein (10 μmol/L), and γ-globulins (80 μmol/L) was assessed, also using the equilibrium dialysis method, to understand which protein(s) were responsible for binding of 8 and M2 D in human plasma. All the test concentrations of Gancao compounds were at 50 μmol/L, except for 27 at 10 μmol/L. Dialysates and associated plasma/protein preparations were sampled for liquid chromatography/mass spectrometry analysis. Rats received in-house femoral artery cannulation for blood sampling or bile duct cannulation for bile sampling, as described previously [13] . A total of 64 rats were used in the following five rat studies.

In the first study, six rats were randomly assigned into two groups to receive orally a dose of LianhuaQingwen at 3.78 g/kg (containing 8 µmol/kg glycyrrhizin) or glycyrrhizin at 8 µmol/kg. The Lianhua-Qingwen dose 3.78 g/kg in rats was nine times the dose translated from the human label daily dose of the capsule (4.2 g/day), using a body surface area normalization method [46] . Serial blood samples (150 μL) were collected before and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 11, 15, 20, and 24 h after dosing and were heparinized and centrifuged to yield plasma samples. This rat study was repeated twice, except for the glycyrrhizin administration group.

In the second study, six rats orally received a dose of LianhuaQingwen at 3.78 g/kg and serial bile samples were collected within 0-1, 1-2, 2-4, 4-6, 6-8, 8-10, 10-24, 24-34, and 34-48 h periods after dosing. In addition, four other rats intravenously received a dose of glycyrrhetic acid at 8 µmol/kg and serial bile samples were collected using the preceding time schedule for LianhuaQingwen. During bile collection, an aqueous solution of sodium taurocholate (7.68 mg/mL; pH 7.4) was infused (1 mL/h) into the duodenum.

In the third study, six rats, housed singly in rat metabolic cages (with urine and fecal collection tubes frozen at −15°C), orally received a dose of LianhuaQingwen at 3.78 g/kg, and serial urine and fecal samples were collected within 0-8, 8-24, 24-32, and 32-48 h periods after dosing. On the day just before the day initiating the excretion study, feces (1-2 g) were freshly collected from each rat and immediately mixed with five-fold volumes of the modified Gifu anaerobic medium broth. After centrifuging at 1000 × g for 5 min, the supernatants containing the colonic microbiota were collected under anaerobic conditions to yield individual samples of RCM. The resulting individual mixtures were combined to yield a pooled preparation of RCM.

In the fourth study, 30 rats orally received a dose of LianhuaQingwen at 3.78 g/kg and killed under isoflurane anesthesia by bleeding from the abdominal aorta at 0, 0.25, 4, 8, and 11 h after dosing (six rats per point time) followed by perfusion with 15 mL saline through the superior vena cava; the blood samples were collected, heparinized, and centrifuged to yield plasma samples. The lungs were excised, rinsed in ice-cold saline, blotted, weighed and homogenized in fivefold (w:w) ice-cold saline.

In the fifth study, six rats, housed singly in rat metabolic cages, intravenously received a dose of glycyrrhetic acid at 8 µmol/kg. Serial blood samples were collected before and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 11, and 24 h after dosing and were heparinized and centrifuged to yield plasma samples, while serial urine samples were also collected within 0-6 and 6-24 h periods.

All the rat study samples were analyzed by liquid chromatography/mass spectrometry.

Composition analysis of LianhuaQingwen for Gancao constituents Composition analysis of LianhuaQingwen for constituents originating from the component Gancao was based on liquid chromatography/mass spectrometry. The analysis was guided by a literature-mined candidate compound list and involved comprehensive detection of constituents present, followed by characterization of the detected constituents and quantification for ranking and grading them. Samples of 29 lots of the capsule were analyzed to evaluate lot-to-lot variability.

To facilitate pharmacokinetic investigation of LianhuaQingwen for compounds originating from Gancao, two types of liquid chromatography/mass spectrometry-based analysis were performed, i.e., (i) profiling of Gancao compounds (unchanged and metabolized) in human/rat study samples that were prepared with methanol and (ii) quantification of selected Gancao compounds in human/rat study samples that were prepared by acidifying with hydrochloric acid and extracting with ethyl acetate and in in vitro metabolism/transport study samples that were prepared with methanol. Although no internal standard was used, assay validation, implemented according to the European Medicines Agency Guideline on bioanalytical method validation (2012; www. ema.europa.eu), demonstrated that the quantification assays developed were reliable and reproducible for the intended use.

Concentrations of hesperetin, 4-methylumbelliferone, midazolam-1'-hydroxylation, chrysin-7-O-glucuronide, flavone-7-O-sulfate, para-aminohippuric acid, prostaglandin F 2α , estrone-3-sulfate, tetraethylammonium, uric acid, glycylsarcosine, estradiol 17β-Dglucuronide, and cortisone in in vitro study samples were measured by liquid chromatography/mass spectrometry.

In vitro assessment of 11β-HSD2 inhibition by Gancao compounds Several Gancao compounds, including glycyrrhetic acid (8), 24hydroxyglycyrrhetic acid (M2 D ), glycyrrhizin (1), licorice saponin G2 (2), glycyrrhetic acid-3-O-glucuronide (7), glycyrrhetic acid-30-Oglucuronide (M8 G ), and liquiritigenin (27) , were assessed for their inhibitory activities on human renal 11β-HSD2, the enzymatic activity of which was indicated by biotransformation of cortisol into cortisone. K m of cortisol for human 11β-HSD2 was measured, using NAD-fortified HKM, to determine the substrate concentration used in inhibition assessment. The inhibition potencies of the test Gancao compounds were initially screened at a concentration of 100 μmol/L and carbenoxolone was used as positive inhibitor [47] . Those Gancao compounds that demonstrated ≥50% inhibition in the initial screening were further evaluated, at multiple concentrations, for their half-maximal inhibitory concentrations (IC 50 ) for 11β-HSD2. For the inhibition assessment, NAD-fortified HKM (after 10-min preincubation at 37°C) were incubated with cortisol at 45 nmol/L (the K m for human 11β-HSD2) for 5 min (under linear biotransformation condition) in the presence or absence of the test Gancao compound. The biotransformation was terminated by mixing with two volumes of ice-cold methanol. After centrifugation at 21100 × g for 10 min, the supernatant was analyzed for cortisone by liquid chromatography/mass spectrometry. This study was repeated but by using NAD-fortified RKM (containing rat renal 11β-HSD2) with cortisol at 450 nmol/L (the K m for the rat enzyme).

Data processing After composition analysis, all the detected and characterized Gancao constituents were ranked in descending order according to their respective daily doses (compound doses) and graded into different levels, i.e., 1-10, 0.1-1, 0.01-0.1, and <0.01 μmol/day. The compound dose was calculated as the product of the compound's content level in LianhuaQingwen and the capsule's label daily dose 4.2 g/day.

Pharmacokinetic parameters of Gancao compounds were estimated by noncompartmental analysis using Kinetica (version 5.0; Thermo Scientific, Philadelphia, PA, USA). Renal clearance ratio (R rc ) was calculated using Eq. 1:

where CL R is the renal clearance, GFR is the glomerular filtration rate (i.e., 107 and 314 mL·h −1 ·kg −1 for humans and rats, respectively [48] ), and f u-plasma is the unbound fraction in plasma. GraFit (version 5.0; Erithacus Software, Surrey, UK) was used to estimate the K m , V max , and IC 50 values.

Statistical analysis was performed using SPSS Statistics software (version 19.0; IBM, Chicago, IL, USA). A value of P < 0.05 was considered to be the minimum level of statistical significance. Table S3) .

Human systemic exposure to Gancao compounds after dosing LianhuaQingwen and their plasma pharmacokinetics and fecal excretion A total of 14 volunteers (male, m1-m8; female, f9-f14) were recruited for pharmacokinetic study of LianhuaQingwen; among them were three male volunteers with appendectomy (m6-m8) and two female volunteers with constipation (f13 and f14; i.e., defecation every 2-3 days). During the human study periods, no serious adverse event was observed in volunteers orally receiving LianhuaQingwen. Urine samples from the volunteer m5 failed to be collected completely, according to the time schedule.

Both unchanged and metabolized Gancao compounds were detected in human plasma samples after dosing LianhuaQingwen (Fig. 3 , Table 1 , and Supplementary Fig. S1 ). Glycyrrhetic acid Table 2 summarizes human pharmacokinetics of glycyrrhetic acid (8) and liquiritigenin (27) in volunteers orally receiving LianhuaQingwen at 12 capsules/day. As shown in Fig. 4(a-d) , plasma concentration-time curves of 8 were unimodal, with peak concentrations occurring 9-24 h (T peak ) and varying lag times of 1-12 h in detection after dosing LianhuaQingwen (except for the male volunteer m1 without such a lag time). The lag times resulted from limited dose of the constituent 8 from dosed LianhuaQingwen; the peak of 8 occurred due to colonic absorption of the microbial metabolite 8 via deglycosylation of unabsorbed constituent glycyrrhizin (1) of the dosed capsule. Significant interindividual differences were observed in level of systemic exposure to 8. Male volunteers with appendectomy (m6-m8) exhibited lower AUC 0-48h values (corrected for body weight) than the other male volunteers (m1-m5) (P < 0.05), while female volunteers with constipation (f13 and f14) exhibited the greatest AUC 0-48h values among all the volunteers. After removing the data of m6-m8, f13, and f14 from comparison, there was no statistically significant gender difference (m1-m5 versus f9-f12) in AUC 0-48h of 8 (P > 0.05). Following repeated administration of LianhuaQingwen for 7 consecutive days, AUC 0-48h values of 8 on day 8 were smaller than the respective values on day 1 (P < 0.05; Table 2 ), i.e., the former being only 16%-67% of the latter, except for f10 being 99%. Notably, renal excretion of 8 after dosing LianhuaQingwen was very slow (CL R , 0.003-0.09 mL·h −1 ·kg −1 ) and involved tubular reabsorption, as indicated by renal clearance ratios (R rc ) of 0.2-0.5. Compared with 8, 24-hydoxyglycyrrhetic acid (M2 D ) exhibited a similar plasma concentration-time profile and a similar profile of interindividual variation in plasma AUC 0-48h (Fig. 4e-h) . Plasma AUC 0-48h of M2 D was only about 17% of that of 8 after dosing the capsule.

Liquiritigenin (27) exhibited bimodal plasma concentrationtime curves, with the first and second peak concentrations occurring 1-3 (T peak-1 ) and 6-12 h (T peak-2 ), respectively, after dosing LianhuaQingwen (Fig. 4i-l) (26) were also detected in the fecal samples, but at low levels.

In vitro metabolism of Gancao compounds To confirm the Gancao metabolites detected in humans after dosing LianhuaQingwen and to facilitate understanding the associated interspecies similarities and differences between humans and rats, individual Gancao compounds were assessed in various in vitro metabolism studies and the results are shown in Fig. 5 and in Supplementary Figs. S2 and S3. Under anaerobic conditions, glycyrrhizin (1) was deglycosylated into glycyrrhetic acid (8) using HCM. Although glycyrrhetic acid-3-O-glucuronide (7) was also detected in the sample at 10 min after initiating incubation, 7 was quickly converted into 8. Meanwhile, licorice saponin G2 (2) was also deglycosylated, using HCM, into the aglycone 24-hydroxyglycyrrhetic acid (M2 D ). Under anaerobic conditions, liquiritin apioside (22) and liquiritin (21) were deglycosylated, using HCM, into their flavonone aglycone liquiritigenin (27) , which was then reduced into davidigenin (M27 R ). Meanwhile, isoliquiritin apioside (23) and isoliquiritin (24) were also deglycosylated into their chalcone aglycone isoliquiritigenin (26) . The conversion of 1 into 8 and those of 22/21 into 27 and then into M27 R were repeatable using RCM under anaerobic conditions. Deglycosylation of glycyrrhizin (1) into glycyrrhetic acid-3-Oglucuronide (7) took place using HLC, but this conversion was significantly slower than such conversion using HCM (Fig. 5) . No glycyrrhetic acid (8) was detected, even when the incubation of 1 with HLC was maintained for 4 and 20 h. Slow deglycosylation of 1 into 7 also took place using RLC.

As shown in Fig. 5 Table 1 . Although the preceding glucuronidation, sulfation, and oxidation of 8 also occurred with RLM and RLC, interspecies differences were observed in enzyme kinetics between the human and rat enzymes (Table 3 and Supplementary Fig. S4 ). In brief, rat hepatic UGT exhibited greater metabolic capability for 8 than human hepatic UGT, while human hepatic SULT exhibited greater metabolic capability than rat hepatic SULT. Formation of M8 O upon incubating 8 with NADPH-fortified human hepatic P450s was significantly poorer than that with rat hepatic P450s. Glucuronidation of liquiritigenin (27) (22); <5 pmol·min −1 ·mg −1 protein]. All these compounds were not substrates of the human renal transporter OAT1, OAT2, OAT3, OCT2, or OCTN1 (responsible for proximal tubular secretion) or OAT4, URAT1, PEPT1, or PEPT2 (responsible for proximal tubular reabsorption), as indicated by the net transport ratios < 3.

Plasma protein binding is an influencing factor of renal excretion via glomerular filtration. As shown in Table 4 , the circulating Gancao compounds glycyrrhetic acid (8) and 24hydroxyglycyrrhetic acid (M2 D ) were extensively bound to human plasma protein, i.e., albumin (rather than α 1 -acid glycoprotein or γglobulins). The compounds related to 8, i.e., glycyrrhizin (1), glycyrrhetic acid-3-O-glucuronide (7), and glycyrrhetic acid-30-Oglucuronide (M8 G ), were also extensively bound to human plasma protein. The circulating Gancao compound liquiritigenin (27) was less bound. Binding profile of rat plasma protein for the preceding compounds was similar to that of human plasma protein (Table 4) . Table 1 and Fig. 3 show Gancao compounds (unchanged and metabolized) that were detected in plasma and/or urine samples of rats orally receiving LianhuaQingwen at 3.78 g/kg. Glycyrrhetic acid [32] [33] [34] [35] [36] [37] [38] [39] , were not detected in the rat plasma or urine samples. Based on interspecies similarities between humans and rats in systemic exposure to and renal excretion of Gancao compounds after dosing LianhuaQingwen and associated interspecies similarities in in vitro metabolism and plasma protein binding, several supportive rat studies were performed to obtain additional pharmacokinetic information on the Gancao compounds, particularly 8.

After orally dosing LianhuaQingwen (at 3.78 g/kg) in rats, glycyrrhetic acid (8), 24-hydroxyglycyrrhetic acid (M2 D ), and liquiritigenin (27) were significantly more abundantly detected in the lungs than other Gancao compounds (Fig. 3) . Lung exposure to Gancao compounds after dosing the capsule appeared to be similar to associated systemic exposure.

A total of 28 Gancao compounds (unchanged and metabolized) were detected in bile samples of rats orally receiving a dose of LianhuaQingwen at 3.78 g/kg ( Fig. 3 and Table 1 ). The biliary Gancao compounds were absorbed Gancao constituents (1-3 and in rats by comparing AUC 0-∞ and C max after an oral dose of the capsule with the respective data after an oral dose of pure glycyrrhizin at the same dose level. The AUC 0-∞ and C max of 1 after dosing LianhuaQingwen were lower than the respective values after dosing pure glycyrrhizin (P < 0.05). However, such pharmacokinetic matrix effects on circulating 8 appeared to be limited ( Supplementary Fig. S5 ).

Inhibitory potency of Gancao compounds on human 11β-HSD2 Using pooled HKM, glycyrrhetic acid (8) and 24hydroxyglycyrrhetic acid (M2 D ) exhibited significantly potent inhibitory activities on human renal 11β-HSD2 compared with the reference compounds glycyrrhizin (1), licorice saponin G2 (2), glycyrrhetic acid-3-O-glucuronide (7) , and glycyrrhetic acid-30-Oglucuronide (M8 G ), as indicated by the IC 50 values (Table 5) . Gancao flavonoids, including liquiritigenin (27) , exhibited poor or negligible inhibition of human 11β-HSD2 (Supplementary Figs. S6  and S7 ). Similar differences were observed using pooled RKM (Table 5 and Supplementary Fig. S7) . Notably, IC 50 values of the test compounds using pooled HKM from Celsis In Vitro Technologies were significantly lower than respective values using pooled HKM from Sekisui XenoTech (Table 5 ). There was no IC 50 value of M2 D for Celsis HKM, because the HKM had run out when the compound was identified in the pharmacokinetic and metabolism studies. Given that only 8 and M2 D were significantly bioavailable for renal 11β-HSD2 with potent inhibitory activities on the enzyme, they were considered to be pseudoaldosterogenic Gancao compounds after dosing LianhuaQingwen.

For a complex Chinese herbal medicine, pharmacokinetic research is a useful approach to identifying its potentially important compounds (i.e., compounds bioavailable at loci responsible for the medicine's therapeutic action) and to characterizing their pharmacokinetics and disposition. Based on clinical use of LianhuaQingwen as treatment for the acute viral respiratory illness, such bioavailability of compounds after dosing the capsule was investigated with respect to their systemic and colon-luminal exposure. A total of 41 Gancao constituents (0.01-8.56 μmol/day) were detected and Table 3 . K m , V max , and CL int data for oxidation, glucuronidation, and sulfation of glycyrrhetic acid (8) . Gancao-induced pseudoaldosteronism is believed to be related to the Gancao saponin constituent glycyrrhizin and its metabolites [31] . However, which Gancao-related compounds are responsible for the adverse effect remains inconclusive, mainly due to incomplete and inaccurate understanding of systemic exposure to glycyrrhizin and its metabolites and their access to renal 11β-HSD2. Furthermore, glycyrrhizin is not the only saponin constituent of Gancao [53, 54] . In the current investigation, Gancao saponin constituents and their metabolites were investigated to identify the compounds that could induce pseudoaldosteronism. The identification was based on human systemic exposure to the Gancao compounds after dosing LianhuaQingwen and their access to and inhibition of renal 11β-HSD2.

Systemic exposure to Gancao saponins after dosing Lianhua-Qingwen is governed by the compounds' metabolism, which mainly comprises two steps: (i) colonic deglycosylation of glycyrrhizin (1) and licorice saponin G2 (2), by colonic microbiota, mainly into absorbable glycyrrhetic acid (8) and 24hydroxyglycyrrhetic acid (M2 D ), respectively, and (ii) hepatic glucuronidation and sulfation of the absorbed 8 and M2 D (Fig. 6) . Despite being the most abundant Gancao saponin constituents in the capsule, unchanged 1 and 2 exhibited low systemic exposure, due to their limited intestinal absorption and their rapid first-pass hepatobiliary excretion [44, 55] . Like 8, glycyrrhetic acid-3-Oglucuronide (7; which had been wrongly considered to be the glucuronide of 8 [33, 34, 36] and responsible for Gancao-induced pseudoaldosteronism [32, 33, 35] ) was a deglycosylated metabolite of 1, but it is not the saponin's major circulating form, due to minor transformation from 1, limited intestinal absorption (due to poor membrane permeability), and rapid deglycosylation into 8. As the respective major circulating forms of 1 and 2, the deglycosylated metabolites 8 and M2 D , were eliminated from general circulation into bile following hepatic conjugations. Glycyrrhetic acid-30-O-glucuronide (M8 G ), rather than 7, was the only glucuronide of 8 (Fig. 5 , Table 1, and Supplementary  Table S5 ). Despite being extensively detected in rat bile, M8 G exhibited low level of systemic exposure in humans and rats receiving LianhuaQingwen, mainly due to poor membrane permeability and weak transport by hepatic MRP3 or MRP4 (Supplementary Table S6 ). Both 8 and M8 G could be sulfated in the liver into glycyrrhetic acid-3-O-sulfate (M8 S ; previously considered as a biomarker of Gancao-induced pseudoaldosteronism [37, 38] ) and glycyrrhetic acid-3-O-sulfate-30-Oglucuronide (M8 G-S ), respectively. Like M8 G , M8 S and M8 G-S were detected in rat bile, but their levels of systemic exposure were low in humans and rats receiving the capsule. Hepatic oxidation of 8 yielded M2 D and a minor hydroxylated metabolite (M8 O ). Both M2 D and M8 O were glucuronized and sulfated, in the liver, into multiple conjugates (M2 D-G , M2 D-S , M2 D-G-S , M8 O-G , M8 O-S , and M8 O-G-S ), all of which could be detected in rat bile samples but not in human and rat plasma samples. Collectively, after dosing LianhuaQingwen, 8 and M2 D were the major circulating Gancao saponin-related compounds; their hepatic conjugated metabolites were mainly excreted into bile, rather than released into the bloodstream.

Renal 11β-HSD2 is highly expressed in the epithelia of the distal tubule and collecting duct [56, 57] . Access of glycyrrhetic acid (8) and 24-hydroxyglycyrrhetic acid (M2 D ) to the renal 11β-HSD2 was evaluated around the renal excretion. Appearance of a drug in the urine is the net result of glomerular filtration, active tubular secretion, and tubular reabsorption. The amount of drug excreted into tubular fluid (urine) via glomerular filtration depends on the systemic exposure level, plasma protein binding, and the GFR. In the proximal tubule, active tubular secretion mediated by basolateral uptake transporters (probably with a joint action of apical efflux transporters) can add the drug into the tubular fluid, while several apical uptake transporters can mediate active tubular reabsorption to reduce the drug in the fluid [58] . In addition, drug having good membrane permeability can undergo passive tubular reabsorption due to concentration gradient for the back-diffusion created by the water reuptake process (particularly in the distal tubule); the degree of the reabsorption is also influenced by the tubular fluid flow and pH (particularly for weak acids and bases with a pK a value of 4.5-8.2). Circulating 8 was excreted into tubular fluid mainly via glomerular filtration, most likely without active tubular secretion and active tubular reabsorption in the proximal tubule. Due to its good membrane permeability, 8 in tubular fluid was reabsorbed into the epithelia by passive diffusion, particularly into the cells of the distal tubule and collecting duct to access 11β-HSD2. The tubular reabsorption of 8 was demonstrated by R rc values of 0.2-0.5 for human volunteers orally receiving LianhuaQingwen and such values of 0.02 for rats intravenously receiving glycyrrhetic acid. Given its good membrane permeability and limited detection in human urine samples after dosing LianhuaQingwen, circulating M2 D is expected to exhibit a similar scenario regarding access to renal 11β-HSD2. Urine pH changes could affect passive tubular reabsorption of 8 and M2 D , (pK a , 4.9; predicted using ACD/ Percepta Platform). Both low systemic exposure and poor membrane permeability limited access of the parent compound glycyrrhizin (1), the conjugated metabolites of 8 and M2 D , and 7 to renal 11β-HSD2. Regarding Gancao-induced pseudoaldosteronism, previous assessment of 11β-HSD2 inhibition by glycyrrhizin and its metabolites by others used the rat enzyme, rather than the human enzyme [35, 39] . Given possible interspecies differences in 11β-HSD2 inhibition [59, 60] , HKM was used to evaluate inhibitory activities of Gancao compounds that were significantly bioavailable at the renal enzyme after dosing LianhuaQingwen. Both 8 and M2 D potently inhibited human renal 11β-HSD2. Notably, significant differences in IC 50 of test compounds were observed when using HKM from different sources (Celsis product versus Sekisui product); these differences may be due to 11β-HSD2 genetic variation. Collectively, the metabolites 8 and M2 D can access and inhibit renal 11β-HSD2, with contribution of 8 to inducing pseudoaldosteronism greater than that of M2 D , mainly due to differences in their systemic exposure levels. Although many conjugated metabolites of 8 and M2 D also inhibit 11β-HSD2, they have limited access to the enzyme.

Excessive and prolonged use of Gancao-containing herbal medicines and dietary products, as well as glycyrrhizin formulations, is a main cause of pseudoaldosteronism [31] . Given the widespread utilization of Gancao products, Gancao-induced pseudoaldosteronism risk during administration of LianhuaQingwen can be potentiated when Gancao-containing dietary products and/or other Gancao-containing medicines are concomitantly ingested. Several factors related to the colon, liver, and kidneys can also influence systemic exposure to glycyrrhetic acid (8) and 24hydroxyglycyrrhetic acid (M2 D ) and/or their access to renal 11β-HSD2. In the colon, deglycosylations of glycyrrhizin (1) and licorice saponin G2 (2) by microbiota and subsequent absorption of the resulting both BCS Class II compounds govern systemic exposure to these two metabolites after dosing LianhuaQingwen. Colonic microbiota exhibits large interindividual variations in composition and function and can be altered by drugs, herbal compounds, and diet, as well as diseases [61, 62] ; this could influence colon-luminal and systemic exposure to 8 and M2 D [63] . Constipation was found to increase plasma AUC 0-48h of 8 and M2 D after dosing LianhuaQingwen, due to, at least in part, the compounds' prolonged colonic absorption. In the liver, the elimination via glucuronidation and sulfation also governs systemic exposure to 8 and M2 D . The hepatic metabolism can be altered by diseases, colonic microbiota, genetic variations of the enzymes, and drug-drug interactions. In the kidneys, glomerular filtration and passive tubular reabsorption, governing access of 8 and M2 D to renal 11β-HSD2, can be affected by changes in overall renal function. Extensive binding to the plasma protein albumin is an important factor limiting the access of 8 and M2 D to renal 11β-HSD2 and in turn contains Gancao-induced pseudoaldosteronism. Accordingly, hepatic synthesis of albumin is important, which could be reduced by hypoalbuminemia (accompanying many diseases, such as chronic hepatitis, cirrhosis, malnutrition, nephrotic syndrome, and sepsis [64] ). SARS-CoV-2 infection can cause gastrointestinal symptoms, such as vomiting, diarrhea, or abdominal pain during the early phases of COVID-19 and induce alterations in intestinal microbiome, as well as in metabolic capabilities of the microbial enzymes [28] . Data on clinical characteristics worldwide indicated that around 60% of COVID-19 positive individuals had one or more preexisting comorbid conditions, such as hypertension, liver diseases, chronic kidney diseases, and metabolic disorders [65] . Given the altered intestinal microbiota and the underlying comorbidity, added precautions should be recommended to prevent Gancao-induced pseudoaldosteronism in such patient populations with comorbidities. To ensure safe use of LianhuaQingwen, (i) increased awareness of Gancaoinduced pseudoaldosteronism and the presence of Gancao in the capsule is required and any pseudoaldosteronism case related to the capsule should be reported; (ii) the capsule should be administered as directed on the leaflets or the prescription and caution should be exercised when the capsule is administered concurrently with other Gancao-containing medicines and dietary products; (iii) caution should be exercised when the capsule is administered in persons with decreased 11β-HSD2 activity, hypokalemia, hypertension, impaired liver function, and hypoalbuminemia; and (iv) once pseudoaldosteronism occurs, the capsule and co-administered Gancao-containing products should be withdrawn, and diuretics (such as spironolactone and eplerenone) and drugs (that alkalinize pH of tubular fluid) may be administered to reduce tubular reabsorption of 8 and M2 D .

In summary, the microbial metabolites glycyrrhetic acid (8), 24hydroxyglycyrrhetic acid (M2 D ; a new Gancao metabolite), liquiritigenin (27) , and glucuronides of 27 (M27 G1 and M27 G2 ) are Gancao compounds circulating significantly in humans receiving LianhuaQingwen. The metabolites 8, M2 D , and 27 are Gancao compounds exhibiting significant colon-luminal exposure, while their respective unabsorbed parent constituents glycyrrhizin (1), licorice saponin G2 (2), and liquiritin/liquiritin apioside (21/22) also have such exposure but with short residence times in the colon. The pharmacokinetics-based identification of these bioavailable Gancao compounds, together with such investigations of other components of LianhuaQingwen, will facilitate uncovering active constituents responsible for the capsule's therapeutic action. In addition, 1 and 2 are metabolically activated by glucuronidase of colonic microbiota to the pseudoaldosterogenic metabolites 8 and M2 D that can access (via passive tubular reabsorption) and potently inhibit renal 11β-HSD2 (with large differences in IC 50 among human kidney microsomes of different sources) (Fig. 8) . Although 8 and M2 D are not the only Gancaorelated compounds exhibiting inhibitory activities on 11β-HSD2, they are the only ones that are bioaccessible for the enzyme. Several factors related to the colon, liver, and kidneys can also influence systemic exposure to 8 and M2 D and/or the compounds' access to renal 11β-HSD2. These findings will help in precisely defining conditions for safe use of LianhuaQingwen and also facilitate safety investigations of other Gancao-containing medicines or herbal products.

Combating COVID-19 with integrated traditional Chinese and Western medicine in China

Chinese National Health Commission and State Administration of Traditional Chinese Medicine. Diagnosis and treatment protocol for novel coronavirus pneumonia

Efficacy and safety of Lianhuaqingwen capsules, a repurposed Chinese herb, in patients with coronavirus disease 2019: a multicenter, prospective, randomized controlled trial

Retrospective clinical analysis on treatment of novel coronavirus infected pneumonia with traditional Chinese medicine Lianhua Qingwen

Analysis of curative effect of 51 patients with novel coronavirus pneumonia treated with Chinese medicine Lianhua Qingwen: a multicentre retrospective study

Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2)

Identifying potential anti-COVID-19 pharmacological components of traditional Chinese medicine Lianhuaqingwen capsule based on human exposure and ACE2 biochromatography screening

Chinese National Health Commission and Chinese State Administration of Traditional Chinese Medicine. Diagnosis and treatment plan for influenza

National Health and Family Planning Commission of People's Republic of China. Gulideline on diagnosis and treatment of Middle East respirotory syndrome (2015 version)

Plasma and urinary tanshinol from Salvia miltiorrhiza (Danshen) can be used as pharmacokinetic markers for cardiotonic pills, a cardiovascular herbal medicine

Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats

Intestinal absorption and presystemic elimination of various chemical constituents present in GBE50 extract, a standardized extract of Ginkgo biloba leaves

Systemic and cerebral exposure to and pharmacokinetics of flavonols and terpenelactones after dosing standardized Ginkgo biloba leaf extracts to rats via different routes of administration

Combinatorial metabolism notably affects human systemic exposure to ginsenosides from orally administered extract of Panax notoginseng roots (Sanqi)

Systemic exposure to and disposition of catechols derived from Salvia miltiorrhiza roots (Danshen) after intravenous dosing DanHong injection in human subjects, rats, and dogs

Pharmacokinetics and disposition of circulating iridoids and organic acids in rats intravenously receiving ReDuNing injection

Pharmacokinetics and disposition of monoterpene glycosides derived from Paeonia lactiflora roots (Chishao) after intravenous dosing of antiseptic XueBiJing injection in human subjects and rats

Pharmacokinetics of catechols in human subjects intravenously receiving XueBiJing injection, an emerging antiseptic herbal medicine

Pharmacokineticsbased identification of potential therapeutic phthalides from XueBiJing, a Chinese herbal injection used in sepsis management

Comparison of intramuscular and intravenous pharmacokinetics of ginsenosides in humans after dosing XueShuanTong, a lyophilized extract of Panax notoginseng roots

Human pharmacokinetics of ginkgo terpene lactones and impact of carboxylation in blood on their plateletactivating factor antagonistic activity

Poly-pharmacokinetic study of a multicomponent herbal medicine in healthy Chinese volunteers

Multiple circulating saponins from intravenous ShenMai inhibit OATP1Bs in vitro: potential joint precipitants of drug interactions

Intravenous formulation of Panax notoginseng root extract: human pharmacokinetics of ginsenosides and potential for perpetrating drug interactions

High degree of pharmacokinetic compatibility exists between the five-herb medicine XueBiJing and antibiotics comedicated in sepsis care

The human intestinal microbiome in health and disease

Respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia

Gastrointestinal symptoms associated with COVID-19: impact on the gut microbiome

Novel coronavirus infection and gastrointestinal tract

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus

Licorice abuse: time to send a warning message

3-Monoglucuronyl-glycyrrhetinic acid is a major metabolite that causes licorice-induced pseudoaldosteronism

A possible involvement of 3-monoglucuronyl-glycyrrhetinic acid, a metabolite of glycyrrhizin (GL), in GL-induced pseudoaldosteronism

Downregulation of a hepatic transporter multidrug resistance-associated protein 2 is involved in alteration of pharmacokinetics of glycyrrhizin and its metabolites in a rat model of chronic liver injury

3-Monoglucuronyl-glycyrrhretinic acid is a substrate of organic anion transporters expressed in tubular epithelial cells and plays important roles in licoriceinduced pseudoaldosteronism by inhibiting 11β-hydroxysteroid dehydrogenase

3-Monoglucuronyl glycyrrhretinic acid is a possible marker compound related to licorice-induced pseudoaldosteronism

18β-Glycyrrhetyl-3-O-sulfate would be a causative agent of licorice-induced pseudoaldosteronism

Identification of glycyrrhizin metabolites in humans and of a potential biomarker of liquorice-induced pseudoaldosteronism: a multi-centre cross-sectional study

Isolation of a novel glycyrrhizin metabolite as a causal candidate compound for pseudoaldosteronism

Defining the role of gut bacteria in the metabolism of deleobuvir: in vitro and in vivo studies

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

Methylation and its role in the disposition of tanshinol, a cardiovascular carboxylic catechol from Salvia miltiorrhiza roots (Danshen)

Renal tubular secretion of tanshinol: molecular mechanisms, impact on its systemic exposure, and propensity for dose-related nephrotoxicity and for renal herb-drug interactions

Glycyrrhizin has a high likelihood to be a victim of drug-drug interactions mediated by hepatic organic aniontransporting polypeptide 1B1/1B3

Rapid and direct measurement of free concentrations of highly protein-bound fluoxetine and its metabolite norfluoxetine in plasma

Dose translation from animal to human studies revisited

Cloning and production of antisera to human placental 11β-hydroxysteroid dehydrogenase type 2

Physiological parameters in laboratory animals and humans

Gut microbiota: a potential new territory for drug targeting

The gut microbiome as therapeutic target

18β-Glycyrrhetinic acid: its core biological properties and dermatological applications

Phytochemical and pharmacological role of Liquiritigenin and isoliquiritigenin from radix glycyrrhizae in human health and disease models

Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice)

Separation and characterization of phenolic compounds and triterpenoid saponins in licorice (Glycyrrhiza uralensis) using mobile phase-dependent reversed-phase × reversed-phase comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

Absorption, disposition, and pharmacokinetics of saponins from Chinese medicinal herbs: what do we know and what do we need to know more?

11β-hydroxysteroid dehydrogenases: intracellular gate-keepers of tissue glucocorticoid action

Glucocorticoids and 11β-hydroxysteroid dehydrogenases: mechanisms for hypertension

Renal transporters in drug development

Inhibition of 11β-hydroxysteroid dehydrogenase 2 by the fungicides itraconazole and posaconazole

Characterization of activity and binding mode of glycyrrhetinic acid derivatives inhibiting 11β-hydroxysteroid dehydrogenase type 2

Extensive impact of non-antibiotic drugs on human gut bacteria

The gut microbiota at the intersection of diet and human health

Pretreatment with broadspectrum antibiotics alters the pharmacokinetics of major constituents of Shaoyao-Gancao decoction in rats after oral administration

Prevalence of comorbidities among individuals with COVID-19: A rapid review of current literature

Competing interests: The authors declare no competing interests.