Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases
Introduction
Amentoflavone (AMF), a natural biflavonoid compound distributed in Selaginella tamariscina, Cupressus funebris, Ginkgo biloba and Hypericum perforatum, possesses a variety of biological activities including anxiolytic, anti-inflammatory, anti-oxidative, anti-cancer, and neuroprotective effects [[1], [2], [3], [4], [5]]. In contrast to the extensive studies on its pharmacological effects, the toxicity and side effects of AMF are rarely reported. Ten years ago, a case report has reported that oral administration of C. funebris extract would trigger acute hepatic failure and acute renal failure, while AMF was the major biflavonoid in this extract [6]. This finding suggests that constitutes from C. funebris may bring some toxic effects, but the underlying mechanism of C. funebris extract induced acute hepatic/renal failure has not been revealed yet. In addition, several studies have reported that AMF can strongly inhibit the catalytic activities of many human cytochrome P450 enzymes (CYPs) including CYP2C9, 2C19, 2D6 and 3A4 [7,8]. These studies suggest that AMF can affect the metabolic elimination of many CYP-substrate drugs or endogenous compounds, which may bring undesirable effects, such as adverse herb-drug interactions (HDIs).
As the most important class of detoxification enzymes, UDP-glucuronosyltransferases (UGTs) play pivotal roles in the metabolic elimination and detoxification of a wide variety of endogenous (e.g., bilirubin and steroids) and xenobiotic (e.g., etoposide, propofol and diethylstilbestrol) compounds. To date, more than twenty human UGTs have been identified, which fall into two major families, i.e., UGT1 and UGT2. Human UGTs are highly expressed in metabolic organs including liver, intestine and kidney, which together constitute the most important part of the metabolic defense system of human body [[20], [21], [22], [23]]. Strong inhibition on human UGTs by xenobiotics may trigger some undesirable effects, including adverse herb/drug-drug interactions (H/DDIs), and some metabolic disorders (such as hyperbilirubinemia and liver disorder) [24,25]. To avoid the potential risks of UGTs-mediated H/DDIs and the occurrence of UGTs-inhibition associated undesirable effects, it is necessary to evaluate the inhibitory effects of drug candidates or major constituents in herbs on UGTs. Taking into account that AMF is an abundant constituent from various medicinal plants, and the inhibitory effects of AMF on human phase II drug metabolizing enzymes (such as UGTs) have not been well-characterized, it is necessary to evaluate the potential inhibitory effects of AMF on UGTs in vitro and to explore AMF-associated toxicity from the perspective of UGTs inhibition.
In the present study, the inhibitory effects of AMF on human UGTs were fully investigated. A panel of commercially available human UGTs was used to screen the inhibition potential of AMF on the glucuronidation activities of each UGT isoform in vitro, using a nonspecific probe substrate (4-methylumbelliferone, 4-MU) for all tested UGTs except for UGT1A4 and 2B10, a specific probe substrate (trifluoperazine, TFP) for UGT1A4, a specific probe substrate (cotinine) for UGT2B10, a specific probe for UGT1A1 (N-3-carboxy propyl-4-hydroxy-1,8-naphthalimide, NCHN) and a specific probe for UGT1A9 (propofol). Furthermore, human liver microsomes (HLMs) were utilized as the enzyme source to investigate the inhibitory effects of AMF on three important human UGTs (UGT1A1, 1A4 and 1A9), which played key roles in glucuronidation of both endogenous compounds and therapeutic drugs. The inhibition constant (Ki) and inhibition mechanisms of AMF against each UGT isoform were also comprehensively characterized.
Section snippets
Chemicals and reagents
Amentoflavone (AMF), magnolol and nilotinib were purchased from Chengdu Pufei De Biotech Co., Ltd (Chengdu, Sichuan, China). 4-Methylumbelliferone (4-MU), 4-MU-β-D-glucuronide (4-MUG), TFP (dihydrochloride salt), propofol, propofol glucuronide (PG), androstrone, and uridine-5′-diphosphoglucuronic acid (UDPGA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cotinine was purchased from Alfa Aesar (Ward Hill, MA). NCHN and NCHN-O-glucuronide (NCHNG) were synthesized by the authors as
Screening of AMF inhibition potentials on human UGTs
The inhibitory effects of AMF (structure is shown in Fig. 1) on 4-MU-O-glucuronidation activity in UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17, on TFP-N-glucuronidation in UGT1A4, and on cotinine-N-glucuronidation in UGT2B10 were evaluated firstly. Three concentrations (1 μM, 10 μM and 100 μM) of AMF were used in the preliminarily screening experiments assays. As shown in Fig. 2, upon addition of 10 μM AMF, the catalytic activities of most human UGTs were strongly inhibited,
Discussion
As the most important class of detoxification enzymes in mammals, UGTs play pivotal roles in the metabolism and detoxification of a variety of endogenous (such as bilirubin, estrogen and steroids) and xenobiotic compounds including drugs and their metabolites, carcinogens and toxicants [14]. Strong inhibitions of human UGTs may decrease the metabolic elimination of both endogenous and xenobiotic substances in vivo, thus increasing plasma concentrations of drugs, toxins and potential carcinogens
Acknowledgements
This work was supported by the by the NSF of China (81773687, 81703606, 81573501, 81473181), the National Key Research and Development Program of China (2017YFC1700200 and 2017YFC1702000), the National S&T Major Projects of China (2017ZX09101004), Shanghai Municipal Science and Technology Committee of Shanghai outstanding academic leaders plan (18XD1403600), State Key Laboratory of Organ Failure Research (201501), and the Doctoral Scientific Research Foundation of Liaoning Province, China (
References (50)
- et al.
Flavonoid-induced acute nephropathy by Cupressus funebris endl (Mourning Cypress)
Am. J. Kidney Dis.
(2006) - et al.
Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity
Food Chem. Toxicol.
(2010) Roles of human UDP-glucuronosyltransferases in clearance and homeostasis of endogenous substrates, and functional implications
Biochem. Pharmacol.
(2015)- et al.
First-pass metabolism via UDP-glucuronosyltransferase: a barrier to oral bioavailability of phenolics
J. Pharmacol. Sci.
(2011) - et al.
An optimized ratiometric fluorescent probe for sensing human UDP-glucuronosyltransferase 1A1 and its biological applications
Biosens. Bioelectron.
(2015) - et al.
Comparison of the inhibitory effects of tolcapone and entacapone against human UDP-glucuronosyltransferases
Toxicol. Appl. Pharmacol.
(2016) - et al.
Assessment of the inhibition potential of Licochalcone A against human UDP-glucuronosyltransferases
Food Chem. Toxicol.
(2016) - et al.
Identification and characterization of naturally occurring inhibitors against UDP-glucuronosyltransferase 1A1 in Fructus Psoraleae (Bu-gu-zhi)
Toxicol. Appl. Pharmacol.
(2015) - et al.
UDP-glucuronosyltransferases and clinical drug-drug interactions
Pharmacol. Ther.
(2005) - et al.
Effects of Japanese herbal medicine, kampo, on human UGT1A1 activity
Drug Metabol. Pharmacokinet.
(2009)
The inhibitory effects of nor-oleanane triterpenoid saponins from Stauntonia brachyanthera towards UDP-glucuronosyltransferases
Fitoterapia
Human UDP-glucuronosyltransferase isoforms involved in bisphenol A glucuronidation
Chemosphere
Isolation and characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids
J. Biol. Chem.
Dietary green and white teas suppress UDP-glucuronosyltransferase UGT2B17 mediated testosterone glucuronidation
Steroids
Development and validation of an ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method for the simultaneous determination of selected flavonoids in Ginkgo biloba
J. Separ. Sci.
Effects of St. John's wort extract and single constituents on stress-induced hyperthermia in mice
Planta Med.
Phenolic compounds from Hypericum perforatum
Planta Med.
Development of an LC-MS method for simultaneous quantitation of amentoflavone and biapigenin, the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood
Phytochem. Anal.
Antifungal effect of amentoflavone derived fromSelaginella tamariscina
Arch Pharm. Res.
Inhibition of human cytochromes P450 by components of Ginkgo biloba
J. Pharm. Pharmacol.
The role of drug metabolizing enzymes in clearance
Expet Opin. Drug Metabol. Toxicol.
Targeted quantitative proteomics for the analysis of 14 UGT1As and-2Bs in human liver using NanoUPLC-MS/MS with selected reaction monitoring
J. Proteome Res.
Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms in liver, intestine, and kidney using nanobore liquid chromatography-tandem mass spectrometry
Anal. Chem.
Novel resveratrol-based substrates for human hepatic, renal, and intestinal UDP-glucuronosyltransferases
Chem. Res. Toxicol.
The prediction of drug-glucuronidation parameters in humans: UDP-glucuronosyltransferase enzyme-selective substrate and inhibitor probes for reaction phenotyping and in vitro-in vivo extrapolation of drug clearance and drug-drug interaction potential
Drug Metab. Rev.
Cited by (36)
Flavonoids and saponins: What have we got or missed?
2023, PhytomedicineFlavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CL<sup>pro</sup>: Inhibition potentials, covalent binding sites and inhibitory mechanisms
2021, International Journal of Biological MacromoleculesCitation Excerpt :Therefore, it is necessary to used more practical approaches (such as structural optimizations or drug delivery technologies) to develop more efficacious agents for combating COVID-19 pandemic. Considering that the naturally occurring flavonoids could be extensively metabolized by UDP-glucuronosyltransferases (UGTs) or other conjugative enzymes in humans [48], AGE could be co-administrated with other herbal medicines containing strong inhibitors against human UGTs, such as Fructus Psoraleae [49], or UGTs inhibitors like amentoflavone [50] and licochalcone A [51], which might improve the in vivo therapeutic effects of AGE against COVID-19. Furthermore, this study also offered several leading compounds and a key warhead for designing and developing more efficacious SARS-CoV-2 3CLpro covalent inhibitors.
A broad-spectrum substrate for the human UDP-glucuronosyltransferases and its use for investigating glucuronidation inhibitors
2021, International Journal of Biological MacromoleculesGlabrone as a specific UGT1A9 probe substrate and its application in discovering the inhibitor glycycoumarin
2021, European Journal of Pharmaceutical SciencesCitation Excerpt :Glucuronidation of the substrates in the pooled HLMs in the absence or presence of selective inhibitors for different UGT isoforms was measured in the incubation systems. The inhibitors and their concentrations were as follows: amentoflavone (AMF, 100 μM) for broad UGTs, nilotinib (NB, 10 μM) for UGT1A1, fluconazole (FLU, 500 μM) for UGT2B7, magnolol (MAG, 10 μM) and niflumic acid (NA, 40 μM) for UGT1A9, hecogenin (HEC, 10 μM) for UGT1A4, glycyrrhetinic acid (GA, 20 μM) for UGT1A3 and 2B7, and protopanaxatriol (PPT, 100 μM) for UGT1A1 and 2B7 (Lv et al., 2017, 2018). The glucuronidation metabolite of glabrone (P1) was prepared by incubating 0.5–1.0 mg/mL purified UGT88D1, 0.3 mM P1 (a total of 4.0 mg was used), and 0.6 mM UDPGA in the buffer solution (50 mM Na2HPO4NaH2PO4, pH 8.0) at 37 °C for 24 h.
- 1
These authors contributed this paper equally.