Elsevier

Chemico-Biological Interactions

Volume 284, 25 March 2018, Pages 48-55
Chemico-Biological Interactions

Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases

https://doi.org/10.1016/j.cbi.2018.02.009Get rights and content

Highlights

  • AMF was a potent broad-spectrum natural biflavone inhibitor of human UGTs.

  • The Ki values for AMF against various human UGTs ranged from 0.29 to 11.51 μM.

  • AMF displayed different inhibition mechanisms against human UGTs.

  • The results provide new sights to the underlying mechanism of C funebris induced hepatic and renal toxicity.

Abstract

Amentoflavone (AMF), an abundant natural biflavonoid found in many medicinal plants, displays various beneficial effects including anti-inflammatory, anti-oxidative and anti-cancer. Despite the extensive studies on pharmacological activities, the toxicity or undesirable effects of AMF are rarely reported. In this study, the inhibitory effects of AMF on human UDP-glucuronosyltransferases (UGTs) were carefully investigated. AMF displayed strong inhibition towards most of human UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4 and 2B17, with the IC50 values ranging from 0.12 μM to 16.81 μM. Inhibition constants (Ki) of AMF against various human UGTs varied from 0.29 μM to 11.51 μM. Further investigation demonstrated that AMF was a noncompetitive inhibitor of UGT1A1 mediated NCHN-O-glucuronidation but functioned as a competitive inhibitor of UGT1A1 mediated 4-MU-O-glucuronidation. In addition, AMF was a competitive inhibitor of UGT1A4 mediated TFP-N-glucuronidation in both UGT1A4 and human liver microsomes, while functioned as a competitive inhibitor of UGT1A9 mediated propofol or 4-MU-O-glucuronidation. These findings demonstrated that AMF was a strong and broad-spectrum natural inhibitor of most human UGTs, which might bring potential risks of herb-drug interactions (HDIs) via UGT inhibition. Additionally, this study provided novel insights into the underlying mechanism of AMF-associated toxicity from the perspective of UGT inhibition.

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 (

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