In Vitro Glucuronidation of Wushanicaritin by Liver

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Sep 19, 2017 - Reaction phenotyping, the relative activity factor (RAF) and activity correlation ..... Aladdin Chemicals (Shanghai, China). All other chemicals ...
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Molecular Sciences Article

In Vitro Glucuronidation of Wushanicaritin by Liver Microsomes, Intestine Microsomes and Expressed Human UDP-Glucuronosyltransferase Enzymes Xiaodan Hong 1,2,3,† , Yuanru Zheng 1,2,† , Zifei Qin 1,2,4, *, Baojian Wu 1,2 , Yi Dai 1,2 , Hao Gao 1,2,3 ID , Zhihong Yao 1,2, * ID , Frank J. Gonzalez 5 and Xinsheng Yao 1,2,3,4 1

2 3 4 5

* †

College of Pharmacy, Jinan University, Guangzhou 510632, China; [email protected] (X.H.); [email protected] (Y.Z.); [email protected] (B.W.); [email protected] (Y.D.); [email protected] (H.G.); [email protected] (X.Y.) Guangdong Provincial Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, China Guangzhou Research and Creativity Biotechnology Co., Ltd., Guangzhou 510663, China Integrated Chinese and Western Medicine Postdoctoral research station, Jinan University, Guangzhou 510632, China Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] Correspondence: [email protected] (Z.Q.); [email protected] or [email protected] (Z.Y.); Tel.: +86-208-522-1767 (Z.Q. & Z.Y.); Fax: +86-208-522-1559 (Z.Q. & Z.Y.) These authors contributed equally to this work.

Received: 13 August 2017; Accepted: 14 September 2017; Published: 19 September 2017

Abstract: Wushanicaritin, a natural polyphenol compound, exerts many biological activities. This study aimed to characterize wushanicaritin glucuronidation by pooled human liver microsomes (HLM), human intestine microsomes and individual uridine diphosphate-glucuronosyltransferase (UGT) enzyme. Glucuronidation rates were determined by incubating wushanicaritin with uridine diphosphoglucuronic acid-supplemented microsomes. Kinetic parameters were derived by appropriate model fitting. Reaction phenotyping, the relative activity factor (RAF) and activity correlation analysis were performed to identify the main UGT isoforms. Wushanicaritin glucuronidation in HLM was efficient with a high CLint (intrinsic clearance) value of 1.25 and 0.69 mL/min/mg for G1 and G2, respectively. UGT1A1 and 1A7 showed the highest activities with the intrinsic clearance (CLint ) values of 1.16 and 0.38 mL/min/mg for G1 and G2, respectively. In addition, G1 was significantly correlated with β-estradiol glucuronidation (r = 0.847; p = 0.0005), while G2 was also correlated with chenodeoxycholic acid glucuronidation (r = 0.638, p = 0.026) in a bank of individual HLMs (n = 12). Based on the RAF approach, UGT1A1 contributed 51.2% for G1, and UGT1A3 contributed 26.0% for G2 in HLM. Moreover, glucuronidation of wushanicaritin by liver microsomes showed marked species difference. Taken together, UGT1A1, 1A3, 1A7, 1A8, 1A9 and 2B7 were identified as the main UGT contributors responsible for wushanicaritin glucuronidation. Keywords: wushanicaritin; glucuronidation; UDP-glucuronosyltransferase; activity correlation analysis; relative activity factor; species difference

1. Introduction Xenobiotic metabolism, a ubiquitous natural response to foreign drugs, elicits initiating signals for many pathophysiological events [1]. According to different drug-metabolizing enzyme systems, xenobiotic biotransformation reactions could be classified to Phase I and Phase II reactions [2,3]. Normally, Phase I reactions included oxidation, reduction and hydrolysis, and cytochrome P450s Int. J. Mol. Sci. 2017, 18, 1983; doi:10.3390/ijms18091983

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enzymes (CYPs) in liver microsomal were the major Phase I metabolic enzymes [4]. The metabolites introduced several reactive functional substituents (–OH, –NH2 , –SH, –H2 ) in metabolic soft spots of the parent drug [5]. Meanwhile, the reactions may produce pharmacologically-active metabolites or toxic metabolites, all of which were very likely to cause variations in drug efficacy [6]. Unlike the Phase I reactions, Phase II reactions always involve combination reactions. The major conjugation reactions were glucuronidation, acetylation, sulfation and methylation. Correspondingly, uridine diphosphate-glucuronosyltransferase (UGT), acetylase, sulfate transferase and methylase were responsible for Phase II catalytic reactions [7,8]. Actually, Phase II reactions were considered as one of the most important detoxification processes due to the obvious increased hydrophilicity of the conjugated metabolites, which could easily promote the excretion of the drug from the body [9,10]. It is well known that glucuronidation is the principal Phase II metabolism because it accounted for the clearance of ~35% drugs metabolized by Phase II enzymes [11]. In human, UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes involved in the glucuronidation of many endogenous and exogenous compounds (e.g., bilirubin, estradiol and polyphenols), especially in many clinical drugs [12,13]. The reaction involves the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to an acceptor substrate to generate more polar and excretable compounds. Human UGT enzymes are classified into families UGT1, UGT2, UGT3 and UGT8 and subdivided into UGT1A, 2A and 2B subfamilies based on gene structure and sequence homology [14]. Traditionally, UGT1A (including nine members) and UGT2B (including seven members) were primarily responsible for the metabolism of xenobiotics or drugs [15]. Usually, the UGT1A and 2B enzymes contributed significantly to metabolism and detoxification of xenobiotics. UGT1A1, 1A3, 1A4, 1A5, 1A6 and 1A9 are expressed in the liver, the major site of glucuronidation, whereas UGT1A7, 1A8 and 1A10 are extrahepatic. All seven UGT2B enzymes (UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28) are found in the liver [16]. Wushanicaritin is a major active compound isolated from the traditional Chinese medicine Epimedium plants. Previous diphenyl picryl hydrazinyl radical (DPPH) radical scavenging activity tests indicated that wushanicaritin (IC50 = 35.3 µM) exhibited significant antioxidant activity comparable to vitamin C (IC50 = 32.0 µM) [17]. In addition, it displayed anti-inflammatory potential in murine macrophage cell lines, as well as in a mouse model of inflammation [18]. Similarly, the anti-inflammatory property of wushanicaritin in human immune cells, especially in monocytes, proved to be mediated, at least partially, via inhibition of the cluster of differentiation 14/toll-like receptor 4 (CD14/TLR4) signaling pathway [19]. Recently, it had been reported that the combination of wushanicaritin and the antiviral drug ganciclovir (GCV) is more effective in inducing extranodal NK/T-cell lymphoma (ENKL) cells’ apoptosis than wushanicaritin or GCV alone, which indicated that wushanicaritin exert significant antitumor effects [20]. So far, the metabolic pathways of wushanicaritin remain unknown. The presence of phenolic functional groups suggested that wushanicaritin may undergo glucuronidation. This knowledge is of great importance for a better understanding of wushanicaritin disposition and its mechanisms of action in vivo. In this study, we aim to characterize the metabolism of wushanicaritin via the glucuronidation pathway and to identify the main UGT enzymes involved in wushanicaritin glucuronidation. The rates of glucuronidation were determined by incubating wushanicaritin with uridine diphosphoglucuronic acid (UDPGA)-supplemented microsomes. Kinetic parameters were derived by fitting an appropriate model to the data. Several series of independent experiments including reaction phenotyping, determination of the relative activity factors (RAF) and activity correlation analyses were performed to identify the main UGT enzymes contributing to the hepatic metabolism of wushanicaritin. It was shown for the first time that wushanicaritin was efficiently metabolized via glucuronidation. Furthermore, UGT1A1, 1A3, 1A7, 1A8, 1A9 and 2B7 were identified as the main contributors to the glucuronidation of wushanicaritin.

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2. Results 2. Results 2.1. Structural Identification of Wushanicaritin Metabolites 2.1. Structural Identification of Wushanicaritin Metabolites After incubation of wushanicaritin with uridine diphosphoglucuronic acid (UDPGA)After incubation wushanicaritin with uridine diphosphoglucuronic (UDPGA)-supplemented supplemented humanofliver microsomes (HLM), two additional peaks (tRacid = 1.93 and 2.53 min), which human liver microsomes (HLM), two additional peaks (t = 1.93 and 2.53 min), which have a similar R have a similar ultraviolet (UV) absorption profile, in addition to wushanicaritin were detected by ultraviolet (UV) absorption profile, in addition to wushanicaritin were detected by ultra-performance ultra-performance liquid chromatography coupled diode-array detector (UPLC-DAD) analysis liquid chromatography coupled diode-array detector (Figure 1a). main Wushanicaritin (Figure 1a). Wushanicaritin exhibited a typical [M +(UPLC-DAD) H]+ ion at m/zanalysis 387.1439 and two daughter + ion at m/z 387.1439 and two main daughter ions at m/z 369.1335 and exhibited a typical [M + H] ions at m/z 369.1335 and 313.0713 produced by losing a neutral fragment of H2O and C4H8, 313.0713 produced losing a neutral fragment H2G2 O and 1b).563.1749, For the 4 Hsame 8 , respectively respectively (Figureby 1b). For the metabolites, G1 of and had C the [M + H]+ (Figure ion at m/z + metabolites, G1 and G2 thethan samethat [M of + H] ion at m/z 563.1749, which wason 176.0325 Da higher than which was 176.0325 Da had higher wushanicaritin (Figure 1b). Based these data, they were that of wushanicaritin (Figure 1b). Based on these data, they were characterized as mono-glucuronides characterized as mono-glucuronides of wushanicaritin. of wushanicaritin. In addition, to determine the exact substitution position of glucuronidation, G1 (about 1 mg) and In addition, to determine the exact substitution glucuronidation, G1 (about(RLM) 1 mg) G2 (less than 1 mg) were biosynthesized and purifiedposition by usingofpooled rat liver microsomes 1 13 and G2 (less than 1 mg) were biosynthesized and purified by using pooled rat liver microsomes and then structurally analyzed by H and C nuclear Magnetic Resonance (NMR) on a Bruker AV13 C nuclear Magnetic (RLM) and then structurally analyzedGermany). by 1 H andThe (NMR) a Bruker 600 spectrometer (Bruker, Newark, signals for the 1H Resonance and 13C (600 MHz,onDMSO-d 6, 1 H and 13 C (600 MHz, DMSO-d , AV-600 spectrometer (Bruker, Newark, Germany). The signals for the 6 298.2 °C) of wushanicaritin and its two glucuronides were assigned and compared with those of 298.2 ◦ C) of wushanicaritin and its two glucuronides wereNMR assigned and compared with thoseS2. of wushanicaritin (Table S1). Meanwhile, their corresponding parameters are shown in Table 1H-NMR 13C-NMR wushanicaritin S1). and Meanwhile, their corresponding parameters in TableThe S2. Similarly, their (Table data are shown inNMR Figures S1 and are S2, shown respectively. 1 H-NMR and 13 C-NMR data are shown in Figures 1 Similarly, their S1 and S2, respectively. The phenolic phenolic proton signals of the C-3 phenolic group in the H-NMR spectrum of G1 and the C-7 1 H-NMR spectrum of G1 and the C-7 phenolic group 1H-NMR proton signals phenolic group in phenolic groupofinthe theC-3 spectrum ofthe G2 both disappeared, respectively. This evidence above 1 in the H-NMR spectrum of G2 both disappeared, respectively. This evidence above indicated that the indicated that the presence of glucuronidation substitutions should be at the C-3 and C-7 phenolic 13 presence of glucuronidation substitutions should be at the C-3 and C-7 phenolic groups. Furthermore, groups. Furthermore, the C-3 signal in the C-NMR spectrum of G1 shifted upfield to δ 135.0 (Table 13 C-NMR spectrum of G1 shifted upfield to δ 135.0 (Table S1) caused by the the caused C-3 signal in the S1) by the glucuronidation of the C-3 phenolic group, while C-2 and C-4 shifted downfield to 13 glucuronidation of the C-3 phenolic group, while C-2the and C-4 shifted to δ1155.4 177.7 δ 155.4 and 177.7 (Table S1), respectively. However, amount of G2downfield was less than mg, and the 13 (Table S1),data respectively. the amount of G2 was less than 1 mg, theG2 C-NMR data was not C-NMR was notHowever, completely obtained. Taken altogether, G1and and were identified as completely obtained. Taken altogether, G1 wushanicaritin-7-O-glucuronide and G2 were identified as wushanicaritin-3-O-glucuronide wushanicaritin-3-O-glucuronide (G1) and (G2), respectively. On the (G1) wushanicaritin-7-O-glucuronide respectively. On the other hand, these result were the otherand hand, these result were the same as(G2), previous studies [21,22]: wushanicaritin-3-O-glucuronide samewushanicaritin-7-O-glucuronide as previous studies [21,22]: wushanicaritin-3-O-glucuronide and were eluted successively and on awushanicaritin-7-O-glucuronide reverse C18 chromatographic were eluted successively on a reverse C18 chromatographic column. column.

Figure 1. Ultra-high chromatography analysis analysis (a) (a) and and MS/MS MS/MS spectrum Figure 1. Ultra-high performance performance liquid liquid chromatography spectrum (b) (b) of of wushanicaritin, wushanicaritin-3-O-glucuronide (G1) and wushanicaritin-7-O-glucuronide (G2). wushanicaritin, wushanicaritin-3-O-glucuronide (G1) and wushanicaritin-7-O-glucuronide (G2). pHLM: pHLM:human pooled liver human liver microsomes; HIM:intestine human intestine microsomes. pooled microsomes; HIM: human microsomes.

2.2. Glucuronidation of Wushanicaritin in Human Liver Microsomes (HLM) and Human Intestine 2.2. Glucuronidation of Wushanicaritin in Human Liver Microsomes (HLM) and Human Intestine Microsomes (HIM) Microsomes (HIM) Kinetic profilingrevealed revealedthat thatthe the formation wushanicaritin-3-O-glucuronide in HLM Kinetic profiling formation of of wushanicaritin-3-O-glucuronide (G1)(G1) in HLM was was well modeled by the substrate inhibition equation (Figure 2a), whereas wushanicaritin-7-Owell modeled by the substrate inhibition equation (Figure 2a), whereas wushanicaritin-7-O-glucuronide glucuronide (G2) classical Michaelis–Menten G2 inintestine human (G2) followed thefollowed classical the Michaelis–Menten kinetics, askinetics, well as as G1well andas G2G1inand human intestine microsomes (HIM) (Figure 2b). The glucuronide formation of G1 and G2 in HLM was microsomes (HIM) (Figure 2b). The glucuronide formation of G1 and G2 in HLM was 1.34 1.34 and and 0.35 nmol/min/mg, respectively. Similarly, the glucuronide formation of G1 and G2 in HIM was

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0.74nmol/min/mg, and 1.34 nmol/min/mg, respectively. Glucuronidation of wushanicaritin waswas efficient 0.35 respectively. Similarly, the glucuronideG1 formation of G1 and in G2HLM in HIM 0.74 (CLint = 1.25 mL/min/mg), following the substrate inhibition kinetics with Kin m HLM valueswas of 1.07 μM. and 1.34 nmol/min/mg, respectively. Glucuronidation G1 of wushanicaritin efficient Glucuronidation G2 of wushanicaritin in HLM had a CL int value of 0.69 mL/min/mg. Similarly, the (CLint = 1.25 mL/min/mg), following the substrate inhibition kinetics with Km values of 1.07 µM. CLint values of G1G2 and in HIM were in 0.18 andhad 0.23amL/min/mg, respectively, whereasSimilarly, the Km values Glucuronidation ofG2 wushanicaritin HLM CLint value of 0.69 mL/min/mg. the ofint G1values and G2 were 4.24 and 5.91 μM, respectively. CL ofin G1HIM andin G2the in Michaelis–Menten HIM were 0.18 andmodel 0.23 mL/min/mg, respectively, whereas theInKaddition, values m the values of HIM G1 ininHLM were 32.99 μM. The detailed of µM, G1 and G2 are listed in Table of G1Kiand G2 in the Michaelis–Menten model wereparameters 4.24 and 5.91 respectively. In addition, 1. Ki values of G1 in HLM were 32.99 µM. The detailed parameters of G1 and G2 are listed in Table 1. the

Figure 2. Cont.

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Figure Figure2.2. Kinetic Kinetic profiles profiles for for glucuronidation glucuronidation of of wushanicaritin wushanicaritin by by various various types types of of microsomes. microsomes. (a) (a) Pooled Pooled human human liver liver microsomes microsomes (HLM); (HLM); (b) (b) pooled pooled human human intestine intestine microsomes microsomes (HIM); (HIM); (c) (c)expressed expressedUGT1A1; UGT1A1; (d) (d)expressed expressedUGT1A3; UGT1A3; (e) (e) expressed expressed UGT1A7; UGT1A7; (f) (f) expressed expressed UGT1A8; UGT1A8; (g) UGT1A9; (h) expressed UGT2B7; in each panel, the insert shows the corresponding (g)expressed expressed UGT1A9; (h) expressed UGT2B7; in each panel,figure the insert figure shows the Eadie–Hofstee plot. All experiments were performed in triplicate. Data are expressed as the mean ± SD. corresponding Eadie–Hofstee plot. All experiments were performed in triplicate. Data are expressed G1: wushanicaritin-3-O-glucuronide; G2: wushanicaritin-7-O-glucuronide. as the mean ± SD. G1: wushanicaritin-3-O-glucuronide; G2: wushanicaritin-7-O-glucuronide. Table Table1.1.Kinetic Kineticparameters parametersofofwushanicaritin wushanicaritinglucuronidation glucuronidationby byHLM, HLM,HIM HIMand andexpressed expressedUGT UGT enzymes (mean ± SD). All experiments were performed in triplicate. enzymes (mean ± SD). All experiments were performed in triplicate. Protein Source Metabolite Metabolite V max Vmax(nmol/min/mg) (nmol/min/mg) KK S50(µM) (μM) Protein Source m mororS50 1.34 ± 0.08 1.07 0.13 G1G1 1.34 ± 0.08 1.07 ±±0.13 0.35 ± 0.01 0.50 0.08 G2G2 0.35 ± 0.01 0.50 ±±0.08 0.74 ± 0.05 4.24 0.75 G1G1 0.74 ± 0.05 4.24 ±±0.75 HIM HIM 1.34 ± 0.10 5.91 1.10 G2G2 1.34 ± 0.10 5.91 ±±1.10 G1 1.13 ± 0.11 0.98 ± 0.20 G1 1.13 ± 0.11 0.98 ± 0.20 UGT1A1 UGT1A1 0.11 ± 0.003 0.68 0.08 G2G2 0.11 ± 0.003 0.68 ±±0.08 G1 0.17 ± 0.003 0.45 ± 0.04 UGT1A3 G1 0.17 ± 0.003 0.45 ± 0.04 G2 0.23 ± 0.007 0.61 ± 0.08 UGT1A3 G2 0.23 ± 0.007 0.61 ± 0.08 G1 0.17 ± 0.003 0.47 ± 0.03 UGT1A7 G1G2 0.17 ± 0.003 0.47 ±±0.03 0.23 ± 0.007 0.61 0.08 UGT1A7 G2 0.23 ± 0.007 0.61 ± 0.08 G1 0.02 ± 0.001 0.38 ± 0.03 UGT1A8 G1G2 0.02 ± 0.001 0.38 ±±0.03 0.07 ± 0.002 0.58 0.07 UGT1A8 G2 0.07 ± 0.002 0.58 ± 0.07 G1 0.08 ± 0.001 0.32 ± 0.03 UGT1A9 G1G2 0.08 ± 0.001 0.32 ±±0.03 0.11 ± 0.003 0.39 0.06 UGT1A9 G2G1 0.11 ± 0.003 0.39 ±±0.06 0.03 ± 0.001 2.34 0.19 UGT2B7 0.01 ± 0.001 5.28 0.98 G1G2 0.03 ± 0.001 2.34 ±±0.19 UGT2B7 G2G1 0.010.22 ± 0.001 5.28 ±±0.98 ± 0.02 1.58 0.25 MkLM 0.21 0.008 0.94 0.08 G1G2 0.22 ±±0.02 1.58 ±±0.25 MkLM G2G1 0.210.34 ± 0.008 0.94 ±±0.08 ± 0.01 0.66 0.06 RLM G2 0.37 ± 0.009 0.45 ± 0.03 G1 0.34 ± 0.01 0.66 ± 0.06 RLM ± 0.01 1.11 0.16 G2G1 0.370.19 ± 0.009 0.45 ±±0.03 DLM G2 0.32 ± 0.02 1.17 ± 0.13 G1 0.19 ± 0.01 1.11 ± 0.16 DLM 0.11 0.004 2.22 0.26 G2G1 0.32 ±±0.02 1.17 ±±0.13 RaLM G2 0.56 ± 0.02 3.29 ± 0.39 G1 0.11 ± 0.004 2.22 ± 0.26 RaLM 0.05 0.002 2.53 0.30 G2G1 0.56 ±±0.02 3.29 ±±0.39 GpLM G2 1.43 ± 0.06 0.93 ± 0.06 G1 0.05 ± 0.002 2.53 ± 0.30 GpLM SI, substrate inhibition model; MM, Michaelis–Menten Notes: G2 1.43 ± 0.06 0.93 ± 0.06 HLM HLM

(μM) KKi i(µM)

CLint int CL (mL/min/mg) (mL/min/mg) 1.25 ± ± 0.17 1.25 0.17 0.69 ± ± 0.11 0.69 0.18 ± ± 0.03 0.18 0.03 0.23 ± ± 0.05 0.23 0.05 1.16 ± 0.27 1.16 ± 0.27 0.16 ± ± 0.02 0.16 0.02 0.36 ± 0.03 0.36 ± 0.03 0.38 ± 0.05 0.38 ± 0.05 0.36 ± 0.03 0.36 0.03 0.38 ± ± 0.05 0.38 ± 0.05 0.05 ± 0.004 0.05 0.12±± 0.004 0.01 0.12 ± 0.01 0.24 ± 0.02 0.24 0.02 0.27 ± ± 0.04 0.27 ±±0.001 0.04 0.01

Model Model

32.990±± 6.717 6.717 SI 32.990 SI N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM 17.150 ± 4.478 SI 17.150 ± 4.478 SI N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. MM N.A. 0.002 0.001 MM N.A. 0.01 ±± 0.001 MM N.A. 0.002 0.001 MM 11.57 ± 2.15 0.14 ± ± 0.03 SI 36.23±± 2.15 5.13 0.23 ± ± 0.02 SI 11.57 0.14 0.03 SI 36.23 0.23 0.02 SI 37.11±± 5.13 5.82 0.52 ± ± 0.05 SI 44.22 ± 5.42 0.81 ± 0.06 SI 37.11 ± 5.82 0.52 ± 0.05 SI 29.09±± 5.42 6.58 0.17 ± ± 0.03 SI 44.22 0.81 0.06 SI 44.14 ± 9.09 0.28 ± 0.03 SI 29.09 ± 6.58 0.17 ± 0.03 SI 0.05 MM 44.14N.A. ± 9.09 0.28 ±±0.006 0.03 SI N.A. 0.17 ± 0.02 MM N.A. 0.05 ± 0.006 MM N.A. 0.02 MM N.A. 0.17 ±±0.002 0.02 MM N.A. 1.54 ± 0.11 MM N.A. 0.02 ± 0.002 MM model; available. N.A.N.A., not1.54 ± 0.11 pHLM: MM

pooledSI,human microsomes; HIM: human intestine microsomes; MkLM: pHLM: monkey liver Notes: substrateliver inhibition model; MM, Michaelis–Menten model; N.A., not available. pooled human liver microsomes; HIM: microsomes; human intestine microsomes; monkeyRaLM: liver microsomes; rat liver microsomes; RLM: rat liver DLM: dog liverMkLM: microsomes; rabbit liverRLM: microsomes; microsomes; DLM: dog liver microsomes; rabbit liver microsomes; GpLM: guinea pig liver microsomes; GpLM: guinea pig liver RaLM: microsomes; G1: wushanicaritin-3-O-glucuronide; G1: wushanicaritin-3-O-glucuronide; G2: wushanicaritin-7-O-glucuronide. G2: wushanicaritin-7-O-glucuronide.

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2.3. Reaction Phenotyping with Expressed UDP-Glucuronosyltransferase (UGT) Enzymes 2.3. Reaction Phenotyping with Expressed UDP-Glucuronosyltransferase (UGT) Enzymes To identify the recombinant UGT enzymes involved in the glucuronidation of wushanicaritin, To identify the UGT recombinant UGT enzymes involved the catalysis glucuronidation of wushanicaritin, twelve expressed enzymes were analyzed for in their activities (expressed as twelve expressed UGT enzymes were analyzed for their catalysis activities (expressed as nmol/min/mg nmol/min/mg protein) at the substrate concentrations of 1.25 μM (Figure 3a) and 20 μM (Figure 3b). protein) at the substrate concentrations 1.25 similar µM (Figure 3a)two andtest 20 µM (Figureconcentrations. 3b). The metabolic The metabolic profiles for G1 and G2ofwere under substrate All profiles for G1were and G2 were similar under two substrateofconcentrations. All experiments were experiments performed in triplicate. Thetest formation G1 and G2 mainly contributed to performed in triplicate. The formation of G1 and G2 mainly contributed to expression of UGT1A1, 1A3, expression of UGT1A1, 1A3, 1A7, 1A8, 1A9 and 2B7 enzymes. The other seven UGT enzymes were 1A7, 1A9ofand enzymes. The otherG1 seven were not capable of thethe production not 1A8, capable the2B7 production toward andUGT G2. enzymes In general, UGT1A1 showed highest toward G1 and G2.activities In general, showed activities for the formation glucuronidation forUGT1A1 the formation ofthe G1,highest while glucuronidation UGT1A7 was mainly responsible for the of G1, while UGT1A7 was mainly responsible for the production of G2. production of G2.

Figure 3. Comparisons of glucuronidation rates (a) 1.25 μM; (b) 20 μM and the intrinsic values Figure 3. Comparisons of glucuronidation rates (a) 1.25 µM; (b) 20 µM and the intrinsic values (CLint ) (CLint) (c) of wushanicaritin by twelve expressed UGT enzymes. All experiments were performed in (c) of wushanicaritin by twelve expressed UGT enzymes. All experiments were performed in triplicate. triplicate. Data are expressed as the mean ± SD. N.D.: not detected. G1: wushanicaritin-3-OData are expressed as the mean ± SD. N.D.: not detected. G1: wushanicaritin-3-O-glucuronide; glucuronide; G2: wushanicaritin-7-O-glucuronide. G2: wushanicaritin-7-O-glucuronide.

2.4. Glucuronidation Kinetics by Recombinant UGT Enzymes 2.4. Glucuronidation Kinetics by Recombinant UGT Enzymes Based on reaction phenotyping results, glucuronidation kinetics of active recombinant UGT Based on reaction phenotyping results, glucuronidation kinetics of active recombinant UGT enzymes were analyzed using a series of substrate concentrations. Obviously, the kinetic profiles of enzymes were analyzed using a series of substrate concentrations. Obviously, the kinetic profiles of G1 G1 by UGT1A1 were well modeled by the substrate inhibition equation (Figure 2c), which was in line by UGT1A1 were well modeled by the substrate inhibition equation (Figure 2c), which was in line with its glucuronidation profiles in HLM (Figure 2a), suggesting that UGT1A1 was indeed the most with its glucuronidation profiles in HLM (Figure 2a), suggesting that UGT1A1 was indeed the most crucial enzyme responsible for hepatic glucuronidation of G1. Except UGT1A1, G1 glucuronidation crucial enzyme responsible for hepatic glucuronidation of G1. Except UGT1A1, G1 glucuronidation of wushanicaritin by UGT1A3 (Figure 2d), 1A9 (Figure 2g) and 2B7 (Figure 2h) enzymes was all well of wushanicaritin by UGT1A3 (Figure 2d), 1A9 (Figure 2g) and 2B7 (Figure 2h) enzymes was all well modeled by Michaelis–Menten kinetics, which did not always followed the same kinetics as HLM. modeled by Michaelis–Menten kinetics, which did not always followed the same kinetics as HLM. For the metabolite G1, the intrinsic clearance (CLint) values by UGT1A1, 1A3, 1A9 and 2B7 were 1.16, For metabolite G1, the intrinsic clearance(Table (CLint )1). values bycontrary, UGT1A1, 1A3, 1A9 and 1.16, 0.36,the 0.24 and 0.01 mL/min/mg, respectively On the the profiles of G22B7 by were UGT1A1 0.36, 0.242c), and1A3 0.01(Figure mL/min/mg, respectively 1). On the contrary, theobviously profiles ofmodeled G2 by UGT1A1 (Figure 2d), 1A9 (Figure 2g)(Table and 2B7 (Figure 2h) were by the (Figure 2c), 1A3 (Figure 2d), 1A9 (Figure 2g) and 2B7 (Figure 2h) were obviously modeled the Michaelis–Menten kinetics, which were all the same as the glucuronidation profiles in by HLM Michaelis–Menten kinetics, which were all the same as the glucuronidation profiles in HLM (Figure 2a). (Figure 2a). For G2, CLint values by UGT1A1, 1A3, 1A9 and 2B7 were 0.16, 0.38, 0.27 and For G2, CLint values by UGT1A1, 1A3, andUGT1A1, 2B7 were1A3, 0.16,1A9 0.38, 0.27 and 0.002 0.002 mL/min/mg, respectively (Table 1).1A9 Hence, and 2B7 were the mL/min/mg, main hepatic respectively (Table 1). Hence, UGT1A1, 1A3, 1A9 and 2B7 were the main hepatic UGT enzymes for UGT enzymes for glucuronidation (G1 and G2) of wushanicaritin (Figure 3c). glucuronidation (G1hand, and G2) of wushanicaritin (Figure 3c). of G1 and G2 both well followed the On the other HIM-mediated glucuronidation On the other hand, HIM-mediated glucuronidation of G1 and G2 1A8 both(Figure well followed the Michaelis–Menten kinetics (Figure 2b). Similarly, UGT1A7 (Figure 2e) and 2f) enzymes Michaelis–Menten kinetics (Figure 2b). Similarly, UGT1A7 (Figure 2e) and 1A8 (Figure 2f) enzymes were both analyzed by the classical Michaelis–Menten kinetics. The CLint values of G1 by UGT1A7 were both enzymes analyzed were by the0.36 classical Michaelis–Menten kinetics. The(Table CLint values of G1 by UGT1A7 and 1A8 and 0.05 mL/min/mg, respectively 1), while UGT1A7and and 1A8 enzymes were 0.36 and 0.05 mL/min/mg, respectively (Table 1), while UGT1A7and 1A8-mediated glucuronidation of G2 had CLint values of 0.38 and 0.12 mL/min/mg, respectively 1A8-mediated glucuronidation of G2 had CL values of 0.38 and 0.12 mL/min/mg, respectively int (Table 1). These results indicated that gastrointestinal enzymes (UGT1A7 and 1A8) also played the (Table 1). These results indicated that gastrointestinal enzymes (UGT1A7 and3c). 1A8) also played the their most most important roles in the glucuronidation of wushanicaritin (Figure Taken together, important roles in the glucuronidation of wushanicaritin (Figure 3c). Taken together, their intrinsic intrinsic clearance (CLint) comparison plot is exhibited in Figure 3c. Kinetic profiles of UGT1A1, 1A3, clearance comparison plot is in exhibited in Figure 3c. Kinetic profiles kinetic of UGT1A1, 1A3, 1A7, int )and 1A7, 1A8,(CL 1A9 2B7 are shown Figure 2c–h, whereas the calculated parameters are displayed in Table 1.

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1A8, 1A9 and 2B7 are shown in Figure 2c–h, whereas the calculated kinetic parameters are displayed in Int.Table J. Mol.1. Sci. 2017, 18, 1983 7 of 16 2.5. 2.5. Contribution Contribution of of UGT1A1, UGT1A1, 1A3, 1A3, 1A9 1A9 and and 2B7 2B7 to to Wushanicaritin WushanicaritinGlucuronidation Glucuronidationin inHLM HLM To the the exactexact contribution of UGT1A1, 1A3, 1A9 and to wushanicaritin Toestimate estimate contribution of UGT1A1, 1A3,2B71A9 and 2B7 to glucuronidation wushanicaritin in HLM, the RAF approach was calculated by the intrinsic clearance (CL ) values of int β-estradiol, int glucuronidation in HLM, the RAF approach was calculated by the intrinsic clearance (CL ) values of chenodeoxycholic acid (CDCA), propofol and zidovudine (AZT) glucuronidation in HLM and β-estradiol, chenodeoxycholic acid (CDCA), propofol and zidovudine (AZT) glucuronidation in relative individual expressed UGT enzyme, The analytical conditionsconditions were adopted HLM and relative individual expressed UGTrespectively. enzyme, respectively. The analytical were based on based previous a result, kineticthe profiles β-estradiol, CDCA, propofol adopted on studies previous[12,23]. studiesAs [12,23]. As the a result, kineticofprofiles of β-estradiol, CDCA, and AZT glucuronidation were modeled by the classical Michaelis–Menten kinetics (Figure 4). propofol and AZT glucuronidation were modeled by the classical Michaelis–Menten kinetics (Figure The derived RAFs for UGT1A1, 1A3, 1A9 and 2B7 were 0.55, 0.48, 0.49 and 1.04, respectively 4). The derived RAFs for UGT1A1, 1A3, 1A9 and 2B7 were 0.55, 0.48, 0.49 and 1.04, respectively (Table (Table The scaled G1 0.642 and (=1.16 G2 were 0.642mL/min/mg (=1.16 × and 0.0.55) 2). The 2). scaled CLint valuesCL ofintG1values and G2ofwere × 0.0.55) 0.09mL/min/mg (=0.16 × 0.55) and 0.09 (=0.16 × 0.55) mL/min/mg for UGT1A1, which represented 51.2% and 12.9%and of 0.69 the mL/min/mg for UGT1A1, which represented 51.2% and 12.9% of the CLint values (1.25 CL values (1.25 and 0.69 mL/min/mg) in HLM. The scaled CL values of G1 and G2 were int mL/min/mg) in HLM. The scaled CLint values of G1 and G2 were 0.17int(= 0.36 × 0.48) mL/min/mg and 0.17 (= 0.36 × (=0.38 ×which 0.48) mL/min/mg UGT1A3, which of represented 0.18 (=0.38 × 0.48) 0.48)mL/min/mg mL/min/mgand for0.18 UGT1A3, represented for 13.8% and 26.0% the total 13.8% and 26.0% of the total glucuronidation activity in HLM. The scaled CL values of G1 and G2 int glucuronidation activity in HLM. The scaled CLint values of G1 and G2 were 0.12 (=0.24 × 0.49) were 0.12 (=0.24 0.49)(=0.27 mL/min/mg and 0.13 (=0.27 × 0.49) mL/min/mg UGT1A9, which was mL/min/mg and×0.13 × 0.49) mL/min/mg for UGT1A9, which was for 9.6% and 19.4% of total 9.6% and 19.4% of total glucuronidation activity in HLM. The scaled CL values of G1 and G2 were int glucuronidation activity in HLM. The scaled CLint values of G1 and G2 were 0.01 (=0.01 × 1.04) 0.01 (=0.01 × and 1.04)0.002 mL/min/mg and 0.002 (=0.002 × mL/min/mg for 1.1% UGT2B7, whichofwas mL/min/mg (=0.002 × 1.04) mL/min/mg for1.04) UGT2B7, which was and 0.3% the 1.1% total and 0.3% of the total glucuronidation activity in HLM. glucuronidation activity in HLM.

Figure 4. Kinetic profiles for β-estradiol (a), chenodeoxycholic acid (CDCA) (b), propofol (c) and Figure 4. Kinetic profiles for β-estradiol (a), chenodeoxycholic acid (CDCA) (b), propofol (c) and zidovudine (AZT) (d) glucuronidation by pooled human liver microsomes (HLM) and individual UGTs zidovudine (AZT) (d) glucuronidation by pooled human liver microsomes (HLM) and individual enzymes; in each panel, the insert figure shows the corresponding Eadie–Hofstee plot. All experiments UGTsperformed enzymes;inintriplicate. each panel, the figureasshows the ± corresponding Eadie–Hofstee plot. All were Data areinsert expressed the mean SD. experiments were performed in triplicate. Data are expressed as the mean ± SD.

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Table Kineticparameters parameters and and relative of of substrate glucuronidation by Table 2.2.Kinetic relative activity activityfactor factor(RAF) (RAF)values values substrate glucuronidation pooled human liver microsomes (HLM) and individual expressed UGT enzyme (mean ± SD). All by pooled human liver microsomes (HLM) and individual expressed UGT enzyme (mean ± SD). experiments were performed in in triplicate. All experiments were performed triplicate. Substrate

Vmax (nmol/min/mg) Km (μM) CLint (μL/min/mg) Model V max (nmol/min/mg) K (µM) CLint (µL/min/mg) Model 0.19 ± 0.09 40.96m± 5.44 4.68 ± 0.66 MM β-estradiol 0.19 ± 0.09 40.96±±4.28 5.44 4.68 ± 0.66 MM 0.21 ± 0.01 25.31 8.45 ± 1.50 MM β-estradiol 0.21 ± 0.01 25.31 ± 4.28 8.45 ± 1.50 MM 0.13 ± 0.003 14.32 ± 1.16 8.86 ± 0.74 MM CDCA 0.13 ± 0.003 14.32±±0.74 1.16 8.86± ± 0.74 MM 0.20 ± 0.003 10.90 18.51 1.28 MM CDCA 0.20 ± 0.003 10.90± ± 0.74 18.51 ± 1.28 MM 0.04 ± 0.001 60.71 9.145 0.63 ± 0.10 MM Propofol 0.04 ± 0.001 60.71 ±±5.91 9.145 0.63 ± 0.10 MM 0.06 ± 0.002 45.91 1.28 ± 0.17 MM Propofol 0.06 45.91±±341.4 5.91 1.28 ± 0.17 MM 1.18±± 0.002 0.53 4359.0 0.27 ± 0.02 MM AZT 1.18 ± 0.53 4359.0 ± 341.4 0.27 ± 0.02 MM 0.42 ± 0.01 1604.0 ± 121.0 0.26 ± 0.02 MM AZT UGT2B7 0.42 ± 0.01 1604.0 ± 121.0 0.26 ± 0.02 MM Notes: MM, Michaelis–Menten model. CDCA: chenodeoxycholic acid; AZT: zidovudine. Substrate

Protein Source Protein Source HLM HLM UGT1A1 UGT1A1 HLM HLM UGT1A3 UGT1A3 HLM HLM UGT1A9 UGT1A9 HLM HLM UGT2B7

RAF

RAF

0.55 0.55

0.48 0.48

0.49 0.49

1.04 1.04

Notes: MM, Michaelis–Menten model. CDCA: chenodeoxycholic acid; AZT: zidovudine.

2.6. Activity Correlation Analysis 2.6. Activity Correlation Analysis As mentioned, glucuronidation activity of β-estradiol in HLM is a well-accepted functional As mentioned, glucuronidation activity of β-estradiol HLM is a well-accepted functional marker marker for UGT1A1 [12]. Glucuronidation activitiesinof individual HLMs (n = 12) toward for UGT1A1 [12].glucuronidation Glucuronidation activities of individual HLMs (n = 12) toward wushanicaritin wushanicaritin and β-estradiol glucuronidation were both determined. It was glucuronidation β-estradiol glucuronidation were both determined. It was shown that wushanicaritin shown that and wushanicaritin 3-O-glucuronidation (G1) and 7-O-glucuronidation (G2) were 3-O-glucuronidation (G1)with and β-estradiol 7-O-glucuronidation (G2) were correlated with β-estradiol significantly correlated glucuronidation withsignificantly correlation factors (r = 0.847, p = 0.0005) glucuronidation correlation factors (r = 0.847, p = 0.0005) = were 0.577,significantly p = 0.049), respectively and (r = 0.577, pwith = 0.049), respectively (Figure 5a,b). Similarly, G1and and(rG2 correlated with CDCA = 0.609, p = 0.036) and (r = 0.638, p = 0.026), (Figure 5c,d). (Figure 5a,b). glucuronidation, Similarly, G1 (r and G2 were significantly correlated withrespectively CDCA glucuronidation, wushanicaritin glucuronidation (G1 and G2) was 5c,d). strongly correlated wushanicaritin with propofol (r Furthermore, = 0.609, p = 0.036) and (r = 0.638, p = 0.026), respectively (Figure Furthermore, glucuronidation, = 0.582, p = 0.047) and (r = 0.611, p =propofol 0.035), respectively (Figure Moreover, glucuronidation (G1(rand G2) was strongly correlated with glucuronidation, (r =5e,f). 0.582, p = 0.047) G1 (rand G2 were also correlated with AZT 5e,f). glucuronidation (r =and 0.407, = 0.189) (r = 0.470, and = 0.611, p = 0.035), respectively (Figure Moreover, G1 G2 pwere also and correlated with p = 0.123), respectively(r(Figure 5g,h). results thatp UGT1A1, 1A9 and (Figure 2B7 enzymes AZT glucuronidation = 0.407, p = The 0.189) andindicated (r = 0.470, = 0.123), 1A3, respectively 5g,h). all results playedindicated a criticalthat role in wushanicaritin glucuronidation and werea the main expressed The UGT1A1, 1A3, 1A9 and 2B7 enzymes all played critical rolehepatic in wushanicaritin UGTs for wushanicaritin glucuronidation. glucuronidation and were the main hepatic expressed UGTs for wushanicaritin glucuronidation.

Figure 5. Cont.

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Figure 5. Correlation analysis between wushanicaritin 3-O-glucuronidation and β-estradiol Figure 5. Correlation analysis between wushanicaritin 3-O-glucuronidation and β-estradiol glucuronidation (a), wushanicaritin 7-O-glucuronidation and β-estradiol glucuronidation (b) in glucuronidation (a), wushanicaritin 7-O-glucuronidation and β-estradiol glucuronidation (b) in a a bank of individual human liver microsomes (n = 12); wushanicaritin 3-O-glucuronidation and bank of individual human liver microsomes (n = 12); wushanicaritin 3-O-glucuronidation and CDCA CDCA glucuronidation (c), wushanicaritin 7-O-glucuronidation and CDCA glucuronidation (d) in glucuronidation (c), wushanicaritin 7-O-glucuronidation and CDCA glucuronidation (d) in a bank of a bank of individual human liver microsomes (n = 12); correlation analysis between wushanicaritin individual human liver microsomes (n = 12); correlation analysis between wushanicaritin 3-O-glucuronidation and propofol glucuronidation (e), wushanicaritin 7-O-glucuronidation and 3-O-glucuronidation and propofol glucuronidation (e), wushanicaritin 7-O-glucuronidation and propofol glucuronidation (f) in a bank of individual human liver microsomes (n = 12); correlation propofol glucuronidation (f) in a bank of individual human liver microsomes (n = 12); correlation analysis between wushanicaritin 3-O-glucuronidation and AZT glucuronidation (g), wushanicaritin analysis between wushanicaritin 3-O-glucuronidation and AZT glucuronidation (g), wushanicaritin 7-O-glucuronidation and AZT glucuronidation (h) in a bank of individual human liver microsomes 7-O-glucuronidation and AZT glucuronidation (h) in a bank of individual human liver microsomes (n = 12). All experiments were performed in triplicate. CDCA: chenodeoxycholic acid; AZT: zidovudine. (n = 12). All experiments were performed in triplicate. CDCA: chenodeoxycholic acid; AZT: G1: wushanicaritin-3-O-glucuronide; G2: wushanicaritin-7-O-glucuronide. zidovudine. G1: wushanicaritin-3-O-glucuronide; G2: wushanicaritin-7-O-glucuronide.

2.7. and GpLM GpLM 2.7.Glucuronidation GlucuronidationofofWushanicaritin Wushanicaritin by by DLM, DLM, RLM, RLM, MkLM, MkLM, RaLM RaLM and The and Km values values were were determined determined for for the the formation formation of of wushanicaritin wushanicaritin The apparent apparent VVmax max and Km glucuronides animal microsomes (Table(Table 1). Wushanicaritin was rapidly glucuronides glucuronidesbyby animal microsomes 1). Wushanicaritin wasconverted rapidly into converted into inglucuronides human andin five types of animal microsomes with high the intrinsic clearance (CL int ) values human and five types of animal microsomes with high the intrinsic clearance (CLintof ) 0.02–1.25 mL/min/mg and 0.17–1.54and mL/min/mg G1 and G2,for respectively. The respectively. Michaelis–Menten values of 0.02–1.25 mL/min/mg 0.17–1.54 for mL/min/mg G1 and G2, The model provided themodel best fit to the glucuronidation of wushanicaritin guinea pigwith liver Michaelis–Menten provided the best fit to thekinetics glucuronidation kinetics ofwith wushanicaritin microsomes (GpLM) (Figure 6e). In contrast, wushanicaritin glucuronidation by monkey guinea pig liver microsomes (GpLM) (Figure 6e). In contrast, wushanicaritin glucuronidationliver by microsomes (MkLM) (Figure (MkLM) 6a), RLM(Figure (Figure 6b), microsomes (DLM) 6c) and rabbit monkey liver microsomes 6a), dog RLMliver (Figure 6b), dog liver (Figure microsomes (DLM) liver microsomes (RaLM) (Figure 6d) all followed substrate inhibition The catalyzation (Figure 6c) and rabbit liver microsomes (RaLM) the (Figure 6d) all followedkinetics. the substrate inhibition efficiencies (reflected by CL values, Figure 6f) for G1 of human and animal microsomes kinetics. The catalyzation efficiencies (reflected by CLint values, Figure 6f) for G1 of human andfollowed animal int the order of HLM (1.25 > RLM (0.52 mL/min/mg) DLM (0.17 mL/min/mg) microsomes followed themL/min/mg) order of HLM (1.25 mL/min/mg) > RLM >(0.52 mL/min/mg) > DLM >(0.17 MkLM (0.14 mL/min/mg) > RaLM (0.05 mL/min/mg) > mL/min/mg) GpLM (0.02 mL/min/mg). Similarly, the mL/min/mg) > MkLM (0.14 mL/min/mg) > RaLM (0.05 > GpLM (0.02 mL/min/mg). Similarly, the intrinsic (CLG2 int) were values for G2 were GpLM (1.54 >mL/min/mg) > RLM (0.81 intrinsic clearance (CLintclearance ) values for GpLM (1.54 mL/min/mg) RLM (0.81 mL/min/mg) HLM (0.69 mL/min/mg) > DLM (0.28 mL/min/mg) > MkLM (0.23 mL/min/mg) > RaLM >mL/min/mg) HLM (0.69 >mL/min/mg) > DLM (0.28 mL/min/mg) > MkLM (0.23 mL/min/mg) RaLM (0.17mL/min/mg). mL/min/mg). Clearly, (0.17 Clearly,there therewas wasaa marked markedspecies speciesdifference difference(up (up toto 60.0-fold) 60.0-fold) in in hepatic hepatic glucuronidationof of wushanicaritin. Furthermore, ratsprobably were probably the for bestwushanicaritin model for glucuronidation wushanicaritin. Furthermore, rats were the best model wushanicaritin glucuronidation studies inits humans due tokinetic its appropriate kinetic parameters (Table glucuronidation studies in humans due to appropriate parameters (Table 1). 1).

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Figure 6. Kinetic profiles profiles for for the the glucuronidation glucuronidationofofwushanicaritin wushanicaritinbybyvarious varioustypes typesofofanimal animal 6. Kinetic microsomes. (a) (a) monkey monkeyliver livermicrosomes microsomes(MkLM); (MkLM);(b) (b)rat ratliver livermicrosomes microsomes(RLM); (RLM);(c)(c)dog dogliver liver microsomes (DLM); (DLM); (d) (d) rabbit rabbit liver liver microsomes microsomes(RaLM); (RaLM);(e) (e)guinea guineapig pigliver livermicrosomes microsomes(GpLM); (GpLM); and the intrinsic intrinsicclearance clearance(CL (CL ) values human liver microsomes animals microsomes intint ) values of of human liver microsomes and and five five animals microsomes (f). (f). In each panel, the insert figure shows the corresponding Eadie–Hofstee plot. All experiments In each panel, the insert figure shows the corresponding Eadie–Hofstee plot. All experiments were were performed in triplicate. are expressed as the mean ± SD. HLM: human liver microsomes; performed in triplicate. DataData are expressed as the mean ± SD. HLM: human liver microsomes; G1: wushanicaritin-3-O-glucuronide; G2: wushanicaritin-7-O-glucuronide. wushanicaritin-7-O-glucuronide.* *compared comparedwith withthe theCL CL intint wushanicaritin-3-O-glucuronide; G2: value 0.01,*** ***pp rabbit > guinea pig. Similarly, the CLint values for G2 were guinea pig > rat > human > dog > monkey > rabbit. Overall, UGT1A1, 1A3, 1A7, 1A8, 1A9 and 2B7

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were the main contributors to the glucuronidation of wushanicaritin. Additionally, glucuronidation of wushanicaritin by liver microsomes showed a marked species difference. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/9/1983/s1. Acknowledgments: This work was supported by the Major Project for the International Cooperation and Exchange of the National Natural Science Foundation of China (Grant No. 81220108028), the National Major Scientific and Program of Introducing Talents of Discipline to Universities (B13038) and the Guangdong Provincial Science and Technology Project (2016B090921005). Author Contributions: Xiaodan Hong, Yuanru Zheng, Zifei Qin and Zhihong Yao conceived of and designed the experiments. Xiaodan Hong, Yuanru Zheng and Zifei Qin performed the experiments. Xiaodan Hong and Baojian Wu analyzed the data. Yuanru Zheng, Yi Dai and Hao Gao contributed reagents and analysis tools. Xiaodan Hong, Zhihong Yao, Frank J. Gonzalez and Xinsheng Yao wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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