Metabolic Activation of Rhein: Insights into the Potential Toxicity

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Metabolic activation of rhein: insights into the potential toxicity induced by rhein-containing herbs Yuan Yuan, Jiyue Zheng, Meiyu Wang, Yuan Li, Jianqing Ruan, and Hongjian Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01872 • Publication Date (Web): 30 Jun 2016 Downloaded from http://pubs.acs.org on July 1, 2016

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Journal of Agricultural and Food Chemistry

Metabolic activation of rhein: insights into the potential toxicity induced by rhein-containing herbs

Yuan Yuan1, Jiyue Zheng, Meiyu Wang, Yuan Li, Jianqing Ruan*, Hongjian Zhang*

1

College of Pharmaceutical Sciences, Soochow University, Suzhou, China

1

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Abstract

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Rhein is a major component of the many medicinal herbs such as rhubarb.

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Despite of wide use, intoxication cases associated with rhein-containing herbs are

4

often reported. The present work aimed to investigate if rhein was subject to

5

metabolic activation leading to toxicity. Upon incubations with different species of

6

liver microsomes, three mono-glucuronides were identified, corresponding to two

7

hydroxyl-glucuronides and one acyl glucuronide via the carboxyl group, respectively.

8

Further study revealed that rhein acyl glucuronide was chemically reactive, and

9

showed cytotoxicity towards hepatocarcinoma cells. In addition, significant species

10

differences in glucuronidation of rhein were observed between laboratory animals and

11

humans. Reaction phenotyping experiments demonstrated that rhein acyl glucuronide

12

was catalyzed predominantly by Uridine 5'-diphospho-glucuronosyltransferase 1A1,

13

1A9 and 2B7. Taken together, the present study confirmed that rhein could be

14

metabolically activated via the formation of acyl glucuronide, especially in human.

15 16

Key words: rhein, metabolic activation, acyl glucuronide

17 18

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INTRODUCTION

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Anthraquinone compound rhein, is widely distributed in many medicinal and

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nutritional plants including Rheum palmatum, Polygonum multiflorum, Cassia

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angustifolia and Cassia occidentalis (1). These rhein-containing herbs were widely

23

used for the anti-inflammatory, antidotal, antipyretic and laxative properties in Asia

24

for centuries in countries of China, Japan, and Korea (2-4). In spite of wide use, the

25

relevant intoxication cases due to the consumption of these herbs were reported

26

continuously (5). Recently, the association between children death and consumption

27

of Cassia occidentalis seeds has been reported (6). The anthraquinones were proved

28

to be the major chemicals in the Cassia occidentalis seeds responsible for producing

29

toxicity and the most cytotoxic moiety in rat primary hepatocytes and

30

hepatocarcinoma cell line (HepG2) among all the anthraquinones was rhein (7).

31

Polygonum multiflorum preparation, which also contained rhein and other

32

anthraquinones, was proved to cause acute liver injury as documented in LiverTox

33

website organized by the National Institutes of Health (8).

34

The toxicity of rhein has been reported to be associated with redox cycling and

35

nucleophilic addition reactions with biomolecules (9-11). Rhein was demonstrated to

36

be able to interfere with a number of mitochondrial functions, such as inhibition of the

37

oxidation of nicotinamide adenine dinucleotide - or flavin adenine dinucleotide

38

-linked substrates and oxidative phosphorylation (12-14). It has been reported that

39

rhein was involved in the nucleophilic addition of thiols leading to a depletion of

40

reduced glutathione (15). In addition, rhein has been found to alter the cytoskeleton by

41

affecting the plasma membrane and intracellular membranes (16, 17).

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Rhein underwent extensive metabolism in the liver, in particular glucuronidation

43

(18, 19) and thus exhibited low oral bioavailability in rats and humans (20). The 3

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major circulating form in the plasma after oral administration of rhein-containing

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products should be the corresponding glucuronides rather than the parent compound.

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However, the toxicity studies were all focused on the parent rhein (21, 22). As a

47

carboxylic anthraquinone, rhein has the potential to be metabolized into rhein acyl

48

glucuronide. It was reported that acyl glucuronides could covalently modify

49

endogenous proteins due to their electrophilic capacity that cause substitution

50

reactions with the nucleophilic groups located on proteins or other macromolecules,

51

and this reaction could ultimately lead to adverse drug toxicities associated with

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carboxylic acid-containing drugs (23).

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Most of the toxicity studies of rhein have been performed using parent

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compound against rat’s primary hepatocytes or other cell lines (21-24). However, the

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toxicity was caused by the parent compound directly or by the metabolites remains

56

unknown. In addition, although a number of studies demonstrated that rhein

57

underwent extensive glucuronidation in the liver (25), the formation of rhein acyl

58

glucuronide and the reactivity of rhein acyl glucuronide were still unknown.

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Therefore, this work was designed to study the metabolic activation of rhein

60

through acyl glucuronide formation and the cytotoxicity of rhein acyl glucuronide. In

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addition, the regioselectivity of rhein glucuronidation by different human Uridine

62

5'-diphospho-glucuronosyltransferases (UGTs) was also conducted to find the major

63

UGTs responsible for the metabolic activation of rhein.

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MATERIALS AND METHODS

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Materials

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Rhein and hyperoside (internal standard, IS) were obtained from Shanghai

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Jiukun International Trade Co., Ltd. (Shanghai, China). Alamethicin, L-Glutathione

68

reduced, D-Saccharic acid 1, 4-lactone monohydrate and formic acid were obtained 4

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from Sigma (St. Louis, MO). Uridine diphosphate glucuronic acid (UDPGA) was

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purchased from Roche (Basel, Switzerland). Dimethyl sulfoxide, disodium hydrogen

71

phosphate and sodium dihydrogen phosphate were purchased from Sinopharm

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Chemical Reagent co., Ltd. (Shanghai, China). Acetonitrile of HPLC (high

73

performance liquid chromatography) grade were purchased from Merck (Darmstadt,

74

Germany). Ultra-water was purified by Hitech Laboratory water purification systems

75

(Shanghai, China).

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Human liver microsomes (HLMs, pooled from 20 different organ donors), mouse,

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dog and monkey liver microsomes and recombinant human UGT isoforms (UGT1A1,

78

UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4,

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UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cells

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were purchased from BD Gentest (Woburn, MA). Pooled Sprague-Dawley rat liver

81

microsomes were prepared using standard methods as described previously (26). The

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research protocol was approved by the animal care and use committee at Soochow

83

University.

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Synthesis and NMR analysis of Rhein Acyl Glucuronide

85

The synthesis of rhein acyl glucuronide was outlined in Figure 1. Condensation

86

of rhein 1 with allyl glucuronate 2 (27) using the procedure developed by Bowkett (28)

87

gave the desired conjugate 3 in 30% yield. The 1H NMR signal of the anomeric

88

proton (δ 5.8, 1H) indicates that compound 3 is the desired β-anomer product (28). To

89

remove the allyl protection, compound 3 was treated with Pd(PPh3)4 and morpholine

90

as reported (28). The deprotect product was precipitated from the solvent during the

91

reaction time as a mophine salt, which was filtered and acidified with Amberlyst A-15

92

(H+) to give the free acyl glucuronide 4. Reaction progress was monitored by

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analytical thin layer chromatography performed on silica gel pre-coated plates. 5

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Column chromatography was prefromed with silica gel (100-200 mesh, Qingdao

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Marine Chemical Inc.,Qingdao,China).. 1H NMR and 13C NMR spectra were obtained

96

on 400 MHz (Varian) spectrometers. Chemical shifts were given in ppm using

97

tetramethylsilane (TMS) as internal standard. Mass spectra were obtained using an

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Agilent 1100 LC/MSD Trap SL version mass spectrometer.

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To a solution of rhein (568 mg, 2.0 mmol) and allyl glucuronate 2 (468 mg, 2.0

100

mmol) in MeCN (20 mL) and THF (10 mL) was added HATU (760 mg, 2.0 mmol)

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and N-methylmorpholine (404 mg, 4.0 mmol). The mixture was stirred at room

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temperature under nitrogen for 8 h, and then quenched by addition of Amberlyst A-15

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(H+, 2 equiv). After evaporation the resulting residue was chromatographed, eluting

104

with 10% Ethanol- dichloromethane to give the title compound 3 as a yellow solid

105

(300 mg, 30%). 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.90 (s, 1H), 7.82 (d, J =

106

7.2 Hz, 1H), 7.74 (d, J = 6.8 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 5.96-5.85 (m, 1H),

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5.74 (d, J = 7.2 Hz, 2H), 5.55 (d, J = 5.2 Hz, 1H), 5.41 (br s, 1H), 5.34 (d, J = 17.6 Hz,

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1H), 5.22 (d, J = 10.4 Hz, 1H), 4.62 (d, J = 4.8 Hz, 2H), 4.10 (d, J = 8.8 Hz, 1H),

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3.53-3.42 (m, 3H).

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To a solution of 3 (200 mg, 0.4 mmol) and Pd(PPh3)4 (46 mg, 0.04 mmol) in

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tetrahydrofuran (5 mL) under nitrogen at 0 °C was added morpholine (72 mg, 0.8

112

mmol). The reaction mixture was stirred at the same temperature for 2 h. The

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morpholine salt of compound 4 was precipitated from the solvent during the reaction

114

time. After filtered and washed with diethyl ether (3 mL x 2), the yellow solid was

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suspended in tetrahydrofuran (5 mL) and Amberlyst A-15 (H+, 50 mg) was added.

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After stirring samples at room temperature for 1 h, the reaction mixture was filtered

117

and concentrated to afford the title compound 4 as a yellow solid (65 mg, 35%). 1H

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NMR (400 MHz, DMSO-d6) δ 11.89 (br s, 2H), 8.19 (s, 1H), 7.92 (s, 1H), 7.84 (dd, J 6

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= 8.0, 8.0 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 5.71-5.63 (m,

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2H), 5.35 (br s, 1H), 3.87 (d, J = 8.8 Hz, 1H), 3.46-3.38 (m, 3H).

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Glucuronidation of Rhein in Liver Microsomes

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Rhein at 25 µM was incubated with 5 mM MgCl2, 20 µg/mL alamethicin, 5 mM

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D-saccharicacid 1,4-lactonein 50 mM phosphate buffer (pH 7.4) in the presence of 1

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mg/mL human liver microsomes, mouse liver microsomes, monkey liver microsomes,

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dog liver microsomes or 1.64 mg/mL rat liver microsomes. The reaction was initiated

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by adding 2 mM UDPGA and kept at 37 °C for 40 min.

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Stability of different Rhein Glucuronides in incubation and sample solution

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Rhein at 100 µM was incubated with 1 mg/mL HLMs for 40 min at the same

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condition described above. The incubation solution was prepared by direct

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centrifugation of the above incubation mixture at 13,000 g for 10 min, while sample

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solution was prepared by adding 3 volume of ice-cold methanol containing 3% formic

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acid before further centrifugation. The corresponding supernatant was transferred into

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new tubes to test the rhein glucuronides stability in incubation and sample solution,

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respectively. Aliquots (40 µL) of the supernatants were further kept at 37 °C for

135

different time intervals. At the time point of 0, 2, 4, 6, 8 and 24 h, 120 µL ice-cold

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methanol containing 3% formic acid and IS was added into the incubation solution to

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quench the degradation.

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In vitro Reactivity of Rhein Acyl Glucuronide with glutathione

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15 µM of RG3 was dissolved in phosphate buffered saline (PBS, 50 mM, pH 7.4)

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in the presence or absence of glutathione (GSH, 10 mM) and kept at 37 °C for

141

different time intervals according to the reference (29). Aliquots of 150 µL incubation

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mixture were taken and added to three volume of ice-cold methanol containing 3%

143

formic acid to quench the reaction. 7

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Glucuronidation Activity of Recombinant Human UGTs towards Rhein

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Glucuronidation activity of rhein in pooled HLMs and 12 recombinant human

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UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17)

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was measured in the aforementioned glucuronidation reaction system which contained

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0.1 mg/mL pooled HLMs or individual recombinant human UGTs instead. All

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reactions were conducted in triplicate at 37 °C for 40 min.

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Kinetics of Rhein Glucuronidation in Pooled HLMs and Recombinant Human

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UGTs

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Kinetics of rhein glucuronidation was determined under the initial rate conditions.

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Briefly, rhein (0.2-100 µM) was incubated with 0.1 mg/mL of pooled HLMs or

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selected individual UGT Supersomes (UGT1A1, 1A7, 1A8, 1A9 and 2B7) in the

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presence of 2 mM UDPGA in a total volume of 0.1 mL at 37 °C for 40 min. Each

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experiment was conducted in triplicate.

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Sample Preparation

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All the glucuronidation reactions were terminated by adding 3 volume of

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ice-cold methanol containing 3% formic acid and the IS. The mixtures were vortexed

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immediately followed by centrifugation at 13,000 g for 10 min. Aliquots of the

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supernatants were subjected to LC-MS/MS. The metabolites and rhein were all

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showed desirable stability in the acidic methanol solution at least for 40 h.

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Cytotoxicity of rhein and rhein acyl glucuronide

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HepG2 cells were obtained from American type culture collection. Cells were

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grown in dulbecco's modified eagle medium supplemented with 10% fetal bovine

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serum and 1% penicillin -streptomycin solution in a humidified atmosphere of 5%

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CO2/95% air at 37 °C. 1 × 104 cells/well (in 120 µL of medium) was transferred to a

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96-well plate. Cells were allowed to attach for 24 h and treated with different 8

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concentrations of rhein and RG3. After 24 h of treatment, 100 µg of 3-

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(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was added

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into each well and incubations continued for 4 h. In the end, the medium was removed

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and the MTT formazan was further dissolved in dimethylsulfoxide. The absorbance

173

was determined at 490 nm by a microplate reader (BioTek, Winooski, VT).

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LC-MS/MS Analysis

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All samples were analyzed by a LC-MS/MS system consisting of an API4000

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Qtrap mass spectrometer equipped with a turbo-V ionization source (Applied

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Biosystems, Foster City, CA, USA), two LC-20AD pumps with a CBM-20A

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controller, DGU-20A solvent degasser and a SIL-20A autosampler (Shimadzu,

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Columbia, MD, USA). The mobile phase consisted of 0.1% formic acid water (A) and

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acetonitrile (B). Samples were analyzed on an AgelaVenusil XBP C18 column (50×2.1

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mm, 3 µm) adopting a gradient elution as follows: 0-0.5 min, 10% B; 0.5-1.5 min,

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10-30% B; 1.5-6.0 min, 30-70% B; 6.0-7.0 min 70-100% B; 7.0-8.0 min 100-10% B;

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8-10 min, 10% B. Negative ionization mode was chosen for sample analysis. The

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MS/MS parameters were set as follows: curtain gas, 30 psi; nebulizer gas (GS1), 55

185

psi; turbo gas (GS2), 55 psi; ion spray voltage, 4500 V; and ion source temperature,

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450 °C. In the selected ion transitions, collision energy and declustering potential

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values were set as -40 and -24 V for rhein; -38 and -14 V for rhein glucuronides; -81

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and -38 for hyperoside (IS), respectively. Ion transitions were monitored as follows:

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rhein, 283.0→239.5; rhein glucuronides, 459.0→283.0; hyperoside, 463.1→300.0.

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For the method validation, intraday (three replicates within a day) and interday

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(three replicates per day over 3 days) variations were measured at two concentration

192

levels to determine the precision and accuracy. Both RG3 and rhein showed good

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stability in the solution of methanol containing 3% formic acid (90% left after 72 hr 9

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stock). The calibration curves were constructed by plotting the peak area ratio of the

195

analyte to the IS. A good linearity (r2 = 0.999) in the concentration range of

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100-25000 nM and 10-5000 nM was obtained for rhein and RG3, respectively. At the

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concentrations tested (rhein: 500, 50000 nM; RG3: 50, 500 nM), intra- and inter-day

198

variations were 1%-11 % and 2%-5% for rhein and RG3 respectively, demonstrating

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an adequate reproducibility in the concentration range analyzed. In addition, great

200

accuracies of 85.3%-105.0% were achieved at these these concentrations examined.

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Data Analysis

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All data were presented as means ± standard deviation (S.D.). Apparent kinetics

203

by UGT Supersomes was obtained by fitting the experimental data with

204

Michaelis-Menten (Eq. (1)) model with nonlinear regression analysis using Prism 5.0

205

(GraphPad Software, CA). Assuming a well-stirred model, the in vitro intrinsic

206

hepatic clearance (CLint) was then calculated with the Eq. (2).

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V = (Vmax× [S])/ (Km + [S]) (1)

208

CLint = Vmax/ Km (2)

209

Where v is the velocity of metabolite formation, Vmax is the maximum velocity, Km is

210

the Michaelis constant defined as the substrate concentration at half of Vmax, [S] is the

211

substrate concentration and CLint is the intrinsic clearance of liver.

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RESULTS

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Glucuronidation of Rhein in Different Liver Microsomes

214

When rhein was incubated with rat, mouse, dog, monkey or human liver

215

microsomes in the presence of UDPGA, three additional peaks with the ion transition

216

of 459.0→283.0 were observed in all incubations (Figure 2A & 2B),which were

217

absent in controls. These three peaks (named as RG1, RG2 and RG3) all showed the

218

molecular ion at m/z 459, 176 mass units higher than that of rhein (m/z 283) (Figure 10

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2C). In addition, the MS2 spectrum showed characteristic [M-GlcA-H]- at m/z 283,

220

corresponding to loss of one molecular of glucuronic acid. As a result, these

221

metabolites were tentatively identified as three mono-glucuronides of rhein,

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corresponding to two mono-glucuronides of two hydroxyl groups and one

223

carboxyl group, respectively. Among these three peaks, RG3 showed the same

224

retention time and mass spectra as those of synthesized rhein acyl glucuronide.

225

Therefore, the structure of RG3 was unambiguously identified as rhein acyl

226

glucuronide. In contrast, RG1 and RG2 were tentatively assigned as rhein hydroxyl

227

glucuronides.

228

The formation rates of three mono-glucuronides varied significantly among

229

different species. As shown in Figure 2B, Rhein acyl glucuronide (RG3) was found as

230

the predominant metabolite accounting for about 60% of total glucuronides in both

231

human and monkey liver microsomes (UV reponse showed similar pattern). However,

232

in rat, mouse and dog liver microsomes, RG3 was the lowest metabolites among three

233

glucuronides.

234

Stability of different Rhein Glucuronides in incubation solution

235

After incubation of rhein with human liver micosomes for 40 min, the protein

236

was removed by centrifugation producing the resulting incubation solution. The

237

stability of three glucuronides of rhein was further investigated for 24 h. As shown in

238

Figure 3A, in the incubation solution, RG1 and RG2 remained constant within 24h.

239

However, RG3 decreased along with time and only 54% ± 0.15% remained in the

240

incubation solution after 24 h.

241

The stability of RG3 was further evaluated using standard solely in PBS buffer.

242

As shown in Figure 3B, RG3 standard was not stable in PBS either and it was rapidly

243

transformed into RG4 in the first two hours. Thereafter, the formed RG4 and 11

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remaining RG3 further transformed into rhein along with time, indicating the

245

instability of RG3.

246

Reactivity of Rhein Acyl Glucuronide with GSH in Vitro

247

Incubation of synthesized RG3 with GSH in phosphate buffer (pH 7.4) at 37 °C

248

resulted in rapid decrease of RG3 as shown in Figure 4A&B. Meanwhile, one peak

249

with ion transition of 459.0→283.0 (RG4) and another one with 445→267 (RGSH) as

250

well as rhein appeared and increase rapidly after incubation. The peak RG4 showed

251

the same ion transition as that of RG3, but with different retention time from RG1,

252

RG2 and RG3, indicating that RG4 might be an isomer of RG3 generated by

253

intramolecular migration. The peak RGSH produced a quasi-molecular ion at m/z

254

445.4 at positive model as shown in Figure 4C, 129 Da less than the molecular ion of

255

rhein GSH conjugate. Neural loss of 129 Da is the characteristic mass pattern of GSH

256

conjugates at positive model, due to the loss of pyroglutamic acid (29), indicating

257

RGSH might be a GSH conjugate. Moreover, the MS2 of RGSH showed product ion

258

at m/z 267 (Figure 4C), which was the [M+H-GSH]

259

These results demonstrated that RGSH was the mono-GSH conjugate of rhein.

260

Therefore, RG3 was proved to be chemically reactive by binding with GSH.

261

Identification of UGT Isoforms Involved in Glucuronidation of Rhein

+

ion of rhein GSH conjugate.

262

The relative activities of 12 recombinant human UGT isoforms (UGT 1A1, 1A3,

263

1A4、1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17) expressed in insect

264

cells were determined in terms of the formation of rhein glucuronides at 25 µM. The

265

formation rates of rhein glucuronides after incubation of rhein with UGT isoforms

266

were shown in Figure 5. UGT1A1, 1A7, 1A8, 1A9 and 1A10 catalyzed RG1

267

formation, and UGT1A1, 1A7, 1A8 and 1A9 showed activity towards RG2 formation.

268

RG3 formation was mediated predominantly by UGT 1A1, 1A9 and 2B7. UGT1A1 12

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showed the highest activity, followed by 1A9 and 2B7, towards RG3 formation. UGT

270

1A8 is the most potent isform mediating RG1 formation followed by UGT 1A1, 1A7

271

and 1A9, while UGT 1A8 showed the highest activity towards RG2 followed by UGT

272

1A9, 1A7 and 1A1. More interestingly, UGT 2B7 showed preference of

273

glucuronidation at the carboxyl group.

274

Kinetics of rhein glucuronidation in HLMs and recombitant UGTs

275

The effects of the concentration of rhein on its glucuronidation activity by HLMs

276

and five individual human UGTs were investigated as shown in Figure 6. The Vmax

277

and Km as well as the Clint, which were calculated from the formations of RG1, RG2

278

or RG3, are summarized in Table 1.

279

The RG3 was the major metabolite of rhein formed in HLMs within the tested

280

substrate concentration range (0.2-100 µM), indicating that HLMs preferentially

281

catalyze glucuronidation of rhein at the carboxyl group, rather than the hydroxyl

282

group. All of RG1, RG2 and RG3 formations by HLMs followed typical

283

Michaelis-Menten kinetics. HLMs exhibited the highest Vmax for the formation of

284

RG3 among the three glucuronides, but similar Km for the formation of RG1, RG2

285

and RG3 (77.4, 52.2 and 50.0 for RG1, RG2 and RG3, respectively). As such, the

286

apparent intrinsic clearance of rhein in HLMs through RG3 was much higher than

287

RG1 and RG2.

288

Within the substrate concentration range tested, only UGT 1A1, 1A9 and 2B7

289

catalyzed RG3 formation from rhein. The carboxyl glucuronidation by UGT 1A1,

290

1A9 and 2B7 followed typical Michaelis-Menten kinetics. UGT 1A1 displayed the

291

highest capacity in converting rhein to RG3 with the lowest Km (14.81 µM) and the

292

highest Vmax, followed by 1A9 (Km 20.86 µM) and 2B7 (Km 66.77 µM).

293

UGT 1A1, 1A7, 1A8 and 1A9 catalyzed both RG1 and RG2 formation from 13

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rhein. The phenolic glucuronation by UGT 1A1, 1A7, 1A8 and 1A9 followed typical

295

Michaelis-Menten kinetics. UGT 1A8 exhibited a highest Vmax for the formation of

296

RG1 and RG2 as shown in Figure 6.

297

Cytotoxicity of rhein acyl glucuronide

298

As showed in Figure 7, obvious cytotoxicity of rhein and RG3 towards HepG2

299

cells was observed at 200 µM. The cytotoxicity effect of RG3 was relatively less than

300

rhein, demonstrating the less direct toxicity of RG3 to HepG2 cells. 200 µM of RG3

301

significantly reduced the viability of HepG2 cells to 30.3%, which might be result

302

from chemical reactivity of RG3 by directly binding with biomacromolecule.

303

DISSCUSION

304

The intoxication cases due to consumption of rhein-containing products in

305

human beings were reported from time to time (30). More seriously, recent studies

306

showed that some cases of children death might be associated with the consumption

307

of Cassia occidentalis seeds and the toxic moieties were proved to be rhein with the

308

highest cytotoxicity followed by emodin, aloe-emodin and other rhubarb

309

anthraquinones (6, 7, 31). It was reported that oral administration of rhubarb might

310

lead to hepatotoxicity and nephrotoxicity in rats (32, 33), and mice (34). However,

311

previous acute and subacute toxicity test, mutagenesis test showed that rhubarb was

312

safe and nontoxic in rats (35). One recent study also found that oral administration of

313

4000 mg/kg/d rhubard extracts for 90 days did not cause subchronic toxicity in

314

Sprague Dawley rats (36). Until now, there is still no agreed conclusion about the

315

toxicity of rhein in human beings and other species.

316

The discrepancy in response to oral administration of rhubarb between rats and

317

human beings might come from the species difference in the metabolic activation of

318

rhein. Our results showed that rhein was mainly metabolized into the acyl glucuronide

319

in liver microsomes from primates (human and monkey) which was chemically 14

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reactive. In contrast, the major metabolites of rhein in liver microsomes from rat,

321

mouse and dog were mono-glucuronides at the hydroxyl groups with no reactivity.

322

Species differences in regioselectivity of glucuronidation of rhein should be attributed

323

to varied hepatic UGTs contents across species (37, 38). Due to the species difference

324

in the metabolic activation of rhein, it may difficult to observe comparative toxic

325

response from laboratory animals as that shown in human beings. As such, the

326

monkey should be the suitable animal model for further studies of bioactivation and

327

toxicity of rhein. It is worthy to note that, in addition to the microsomal enzyme

328

activity, other factors, such as organ blood flow, total organ mass, also differ between

329

species and contribute to species difference in vivo.

330

Rhein underwent extensive phase two metabolism in the liver, especially

331

glucuronidation. It was reported that, the majority of rhein is eliminated in urine as

332

glucuronide conjugates (60%), followed by unchanged form (20%) and sulfate

333

conjugates (20%) after oral administration of rhubarb extract to rats (25). Based on

334

clinical reports, the Cmax of rhein in patients administered Rheum sp. Ranged from 6.7

335

to 38.7 mM (39-40), indicating similar or higher exposure level of rhein glucuronides

336

in human beings. In present study, the major glucuronide generated in HLMs was

337

proved to be the rhein acyl glucuronide. As a result, the majority of rhein might be

338

transformed to the acyl glucuronide in the liver, which might be activated and

339

covalently bind with proteins or DNAs and cause toxicity ultimately.

340

For the first time, the rhein acyl glucuronide was proved to be chemically

341

reactive by our study. Rhein acyl glucuronide was not stable in PBS buffer at

342

physiological temperature. It was transferred to an isomer by intramolecular migration.

343

More importantly, rhein acyl glucuronide was capable to covalently bind with GSH to

344

form rhein-GSH adduct, indicating its chemical reactivity. Therefore, rhein might 15

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345

induce toxicity by metabolic activation to rhein acyl glucuronide and further covalent

346

binding with endogenous biomacromolecules (42, 43).

347

For the first time, the circulating metabolite of rhein in the blood (RG3) was

348

proved to be toxic towards HepG2 cells. Previous cytotoxicity studies all focused on

349

the direct toxicity of rhein and neglected the potential toxicity of the metabolites,

350

which might be the main circulating form in the blood. Both direct toxic effects and

351

immune-mediated toxicity (hypersensitivity reactions) might be the possible

352

mechanisms of liver injury caused by acyl glucuronides (44). With direct toxicity,

353

covalent protein binding via acyl glucuronides may disrupt the normal physiological

354

function of a critical protein and /or DNA or some critical regulatory pathway, leading

355

to cellular necrosis. Alternatively, the modified protein bound with chemical reactive

356

acyl glucuronides can act as haptens and initiate an immune reaction that may be

357

mediated via a specific humoral (antibody) response, a cellular response (T

358

lymphocytes), or a combination of both (45, 46). Our result revealed that RG3 showed

359

a relatively lower cytotoxic effect than rhein on HepG2 cell, indicating direct

360

cytotoxicity might not be the major reason of rhein causing toxicity in human beings.

361

Although with less direct cytotoxicity than rhein, the activated rhein acyl glucuronides

362

might still play important role in the toxicity caused by rhein in consideration of the

363

extensive metabolism and potential immune-related toxicity.

364

UGT catalyzed the conjugation of lipophilic compounds in all living organisms.

365

There are 4 families of UGTs expressed in humans and other rodents: UGT1, UGT2,

366

UGT3 and UGT8, among which UGT2B7 is the most abundant UGT in human liver

367

(47). Liver appears to be the major site of UGT distribution, but some UGT those

368

isoforms found only in extrahepatic tissues, such as UGT1A7, 1A8 and 1A10 (48).

369

Our study revealed that the activity and positional preference of glucuronidation of 16

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rhein varied among different UGT isoforms. RG3 formation was mediated

371

predominantly by UGT 1A1, 1A9 and 2B7 and UGT1A1 showed the highest activity,

372

followed by 1A9 and 2B7, towards RG3 formation. Thus, these UGTs may play an

373

important role in both hepatic first-pass elimination of rhein in human body. UGT

374

1A8 is the most potent isform mediating both RG1 and RG2 formation, which

375

specifically expressed in the gastrointestinal tract, indicating that the phenolic

376

hydroxyl groups of rhein predominantly underwent glucuronidation in the

377

gastrointestinal tract. It is worthy to note that UGT 2B7 showed the ability to solely

378

catalyse the glucuronidation of the carboxyl group in rhein, advising the advantage of

379

using UGT 2B7 expressed cell to further investigate the metabolic activation of rhein.

380

In conclusion, the present study confirmed that rhein is metabolized to acyl

381

glucuronide in human beings and the rhein acyl glucuronide is chemically reactive

382

and cytotoxic. There are significant species differences in the activation of rhein

383

between laboratory animals and human beings. The metabolic activation of rhein to its

384

acyl glucuronide might be the underlying mechanism of toxicity caused by

385

rhein-containing products.

386

ABBREVIATIONS

387

HepG2, hepatocarcinoma cell line; UGT, UDP glucuronosyltransferase; UDPGA,

388

uridine

389

chromatography; HLM, human liver microsomes; TMS, tetramethylsilane; GSH,

390

glutathione;

391

bromide; IS, internal standard; PBS, phosphate buffer; RG1 and RG2, rhein hydroxyl

392

glucuronide; RG3, rhein acyl glucuronide

393

AUTHOR INFORMATION

394

Corresponding Author

5’-diphosphoglucuronic

MTT,

acid;

HPLC,

high

performance

liquid

3- ( 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

17

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Journal of Agricultural and Food Chemistry

395

Dr. Jianqing Ruan, Soochow University, College of Pharmaceutical Sciences, Suzhou,

396

215123, People’s Republic of China. (Tel: +86 512 65882089; Fax: +86 512 65882089;

397

E-mail: [email protected])

398

Dr. Hongjian Zhang, Soochow University, College of Pharmaceutical Sciences, Suzhou,

399

215123, People’s Republic of China. (Tel: +86 512 65882659; Fax: +86 512 65882089;

400

E-mail: [email protected])

401

Notes

402

The authors declare no competing financial interest.

403

Funding

404

The present studies were supported by Jiangsu Province Science Foundation for

405

Youths (BK20150349).

18

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406

References

407

1. Dregus, M.; Engel, K. H., Volatile constituents of uncooked rhubarb (Rheum

408

rhabarbarum L.) stalks. J. Agric. Food Chem. 2003, 51, 6530-6536.

409

2. Wang, L.; Pan, X.; Sweet, D. H., The anthraquinone drug rhein potently interferes

410

with organic anion transporter-mediated renal elimination. Biochem. Pharmacol.

411

2013, 86, 991-996.

412

3. Kim, Y. M.; Lee, C. H.; Kim, H. G.; Lee, H. S., Anthraquinones isolated from Cassia

413

tora (Leguminosae) seed show an antifungal property against phytopathogenic fungi.

414

J. Agric. Food Chem. 2004, 52, 6096-6100.

415

4. Cai, Y.; Sun, M.; Xing, J.; Corke, H., Antioxidant phenolic constituents in roots of

416

Rheum officinale and Rubia cordifolia: structure-radical scavenging activity

417

relationships. J. Agric. Food Chem. 2004, 52, 7884-7890.

418

5. Fang, F.; Wang, J. B.; Zhao, Y. L.; Jin, C.; Kong, W. J.; Zhao, H. P.; Wang, H. J.; Xiao,

419

X. H., A comparative study on the tissue distributions of rhubarb anthraquinones in

420

normal and CCl4-injured rats orally administered rhubarb extract. J. Ethnopharmacol.

421

2011, 137, 1492-1497.

422

6. Panigrahi, G.; Tiwari, S.; Ansari, K. M.; Chaturvedi, R. K.; Khanna, V. K.; Chaudhari,

423

B. P.; Vashistha, V. M.; Raisuddin, S.; Das, M., Association between children death and

424

consumption of Cassia occidentalis seeds: clinical and experimental investigations.

425

Food Chem. Toxicol. 2014, 67, 236-248.

426

7. Panigrahi, G. K.; Ch, R.; Mudiam, M. K.; Vashishtha, V. M.; Raisuddin, S.; Das, M.,

427

Activity-guided chemo toxic profiling of Cassia occidentalis (CO) seeds: detection of

428

toxic compounds in body fluids of CO-exposed patients and experimental rats. Chem.

429

Res. Toxicol. 2015, 28, 1120-1132. 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Institutes

430

8. National

431

http://livertox.nih.gov/ShouWuPian.htm

432

9. Ollinger, K.; Brunmark, A., Effect of hydroxy substituent position on

433

1,4-naphthoquinone toxicity to rat hepatocytes. J. Biol. Chem. 1991, 266,

434

21496-21503.

435

10. Powis, G., Free radical formation by antitumor quinones. Free Radic. Biol. Med.

436

1989, 6, 63-101.

437

11. Bousquet, A.; Ceccato, M.; Hinge, M.; Pedersen, S. U.; Daasbjerg, K., Redox

438

grafting of diazotated anthraquinone as a means of forming thick conducting organic

439

films. Langmuir. 2012, 28, 1267-1275.

440

12. Kean, E. A.; Gutman, M.; Singer, T. P., Studies on the respiratory chain-linked

441

nicotinamide adenine dinucleotide dehydrogenase. XXII. Rhein, a competitive

442

inhibitor of the dehydrogenase. J. Biol. Chem. 1971, 246, 2346-2353.

443

13. Kean, E. A.; Gutman, M.; Singer, T. P., Rhein, a selective inhibitor of the

444

DPNH-flavin step in mitochondrial electron transport. Biochem. Biophys. Res.

445

Commun. 1970, 40, 1507-1513.

446

14. Floridi, A.; Castiglione, S.; Bianchi, C., Sites of inhibition of mitochondrial electron

447

transport by rhein. Biochem. Pharmacol. 1989, 38, 743-751.

448

15. Bellomo, G.; Mirabelli, F.; DiMonte, D.; Richelmi, P.; Thor, H.; Orrenius, C.;

449

Orrenius, S., Formation and reduction of glutathione-protein mixed disulfides during

450

oxidative

451

(2-methyl-1,4-naphthoquinone). Biochem. Pharmacol. 1987, 36, 1313-1320.

452

16. Iosi, F.; Santini, M. T.; Malorni, W., Membrane and cytoskeleton are intracellular

453

targets of rhein in A431 cells. Anticancer Res. 1993, 13, 545-554.

stress.

A

of

study

Health,

with

National

Page 20 of 34

isolated

Library

hepatocytes

20

ACS Paragon Plus Environment

of

and

Medicine.

menadione

Page 21 of 34

Journal of Agricultural and Food Chemistry

454

17. Atheer, A.; Anees, A.; Md., Zafaryab; Md., Irshad; Irfan, Ahmad; Zakia, Kazim; Ayaz

455

Ahmad; M. Moshahid A., Rhein induced cell death and apoptosis through caspase

456

dependent and associated with modulation of p53, bcl-2/bax ratio in human cell lines.

457

Int. J. Pharm. Pharm. Sci. 2014, 6, 515-519.

458

18. Dahms, M.; Lotz, R.; Lang, W.; Renner, U.; Bayer, E.; Spahn-Langguth, H.,

459

Elucidation of phase I and phase II metabolic pathways of rhein: species differences

460

and their potential relevance. Drug Metab. Dispos. 1997, 25, 442-452.

461

19. Wu, W.; Hu, N.; Zhang, Q.; Li, Y.; Li, P.; Yan, R.; Wang, Y., In vitro glucuronidation

462

of five rhubarb anthraquinones by intestinal and liver microsomes from humans and

463

rats. Chem. Biol. Interact. 2014, 219, 18-27.

464

20. Wu, W.; Yan, R.; Yao, M.; Zhan, Y.; Wang, Y., Pharmacokinetics of anthraquinones

465

in rat plasma after oral administration of a rhubarb extract. Biomed. Chromatogr.

466

2014, 28, 564-572.

467

21. Bironaite, D.; Ollinger, K., The hepatotoxicity of rhein involves impairment of

468

mitochondrial functions. Chem. Biol. Interact. 1997, 103, 35-50.

469

22. Kagedal, K.; Bironaite, D.; Ollinger, K., Anthraquinone cytotoxicity and apoptosis

470

in primary cultures of rat hepatocytes. Free Radic. Res. 1999, 31, 419-428.

471

23. Miyashita, T.; Kimura, K.; Fukami, T.; Nakajima, M.; Yokoi, T., Evaluation and

472

mechanistic analysis of the cytotoxicity of the acyl glucuronide of nonsteroidal

473

anti-inflammatory drugs. Drug Metab. Dispos. 2014, 42, 1-8.

474

24. Ye, L.; Lu, L.; Li, Y.; Zeng, S.; Yang, X.; Chen, W.; Feng, Q.; Liu, W.; Tang, L.; Liu, Z.,

475

Potential role of ATP-binding cassette transporters in the intestinal transport of rhein.

476

Food Chem. Toxicol. 2013, 58, 301-305.

477

25. Nicolas, P.; Tod, M.; Padoin, C.; Petitjean, O., Clinical pharmacokinetics of 21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

478

diacerein. Clin. Pharmacokinet. 1998, 35, 347-359.

479

26. Pearce, R. E.; McIntyre, C. J.; Madan, A.; Sanzgiri, U.; Draper, A. J.; Bullock, P. L.;

480

Cook, D. C.; Burton, L. A.; Latham, J.; Nevins, C.; Parkinson, A., Effects of freezing,

481

thawing, and storing human liver microsomes on cytochrome P450 activity. Arch.

482

Biochem. Biophys. 1996, 331, 145-169.

483

27. El Alaoui, A.; Schmidt, F.; Monneret, C.; Florent, J. C., Protecting groups for

484

glucuronic acid: application to the synthesis of new paclitaxel (taxol) derivatives. J.

485

Org. Chem. 2006, 71, 9628-9636.

486

28. Bowkett, E. R.; Harding, J. R.; Maggs, J. L.; Park, B. K.; Perrie, J. A.; Stachulski, A. V.,

487

Efficient synthesis of 1 beta-O-acyl glucuronides via selective acylation of allyl or

488

benzyl D-glucuronate. Tetrahedron. 2007, 63, 7596-7605.

489

29. Baillie, T. A.; Davis, M. R., Mass spectrometry in the analysis of glutathione

490

conjugates. Biol. Mass Spectrom. 1993, 22, 319-325.

491

30. Ueo, H.; Motohashi, H.; Katsura, T.; Inui, K., Human organic anion transporter

492

hOAT3 is a potent transporter of cephalosporin antibiotics, in comparison with

493

hOAT1. Biochem. Pharmacol. 2005, 70, 1104-1113.

494

31. Panigrahi, G. K.; Yadav, A.; Srivastava, A.; Tripathi, A.; Raisuddin, S.; Das, M.,

495

Mechanism of rhein-induced apoptosis in rat primary hepatocytes: beneficial effect

496

of cyclosporine A. Chem. Res. Toxicol. 2015, 28, 1133-1143.

497

32. Wang, Y. H.; Zhao, H. P.; Wang, J. B.; Zhao, Y. L.; Xiao, X. H., [Study on

498

dosage-toxicity/efficacy relationship of prepared rhubarb on basis of symptom-based

499

prescription theory]. Zhongguo Zhong Yao Za Zhi. 2014, 39, 2918-2923.

500

33. Yan, M.; Zhang, L. Y.; Sun, L. X.; Jiang, Z. Z.; Xiao, X. H., Nephrotoxicity study of

501

total rhubarb anthraquinones on Sprague Dawley rats using DNA microarrays. J. 22

ACS Paragon Plus Environment

Page 22 of 34

Page 23 of 34

Journal of Agricultural and Food Chemistry

502

Ethnopharmacol. 2006, 107, 308-311.

503

34. Wang, J. B.; Ma, Y. G.; Zhang, P.; Jin, C.; Sun, Y. Q.; Xiao, X. H.; Zhao, Y. L.; Zhou, C.

504

P., Effect of processing on the chemical contents and hepatic and renal toxicity of

505

rhubarb studied by canonical correlation analysis. Acta Pharm. Sin. B. 2009, 44,

506

885-890.

507

35. Donghai Jiao, S. D., Rhubarb research[M]. Shanghai Scientific and Technical

508

Publishers: 2000; p 252-255.

509

36. Ye, B. G.; Feng, Y.; Wang, S., Scientific evaluation of the acute toxicity and

510

13-week subchronic toxicity of Rheum emodi rhizome extracts in Sprague Dawley

511

rats. Food Chem. Toxicol. 2014, 66, 278-285.

512

37. Ohno, S.; Nakajin, S., Determination of mRNA expression of human

513

UDP-glucuronosyltransferases and application for localization in various human

514

tissues by real-time reverse transcriptase-polymerase chain reaction. Drug Metab.

515

Dispos. 2009, 37, 32-40.

516

38. Shelby, M. K.; Cherrington, N. J.; Vansell, N. R.; Klaassen, C. D., Tissue mRNA

517

expression of the rat UDP-glucuronosyltransferase gene family. Drug Metab. Dispos.

518

2003, 31, 326-333.

519

39. Jiang, J. Y.; Yang, M. W.; Qian, W.; Lin, H.; Geng, Y.; Zhou, Z. Q.; Xiao, D. W.,

520

Quantitative

521

chromatography-negative electrospray ionization tandem mass/mass spectrometry

522

and the application in a pharmacokinetic study. J. Pharm. Biomed. Anal. 2012, 57,

523

19-25.

524

40. Zhu, W.; Wang, X. M.; Zhang, L.; Li, X. Y.; Wang, B. X., Pharmacokinetic of rhein in

525

healthy male volunteers following oral and retention enema administration of

determination

of

rhein

in

human

23

ACS Paragon Plus Environment

plasma

by

liquid

Journal of Agricultural and Food Chemistry

526

rhubarb extract: a single dose study. Am. J. Chin. Med. 2005, 33, 839-850.

527

41. Lee, J. H.; Kim, J. M.; Kim, C., Pharmacokinetic analysis of rhein in Rheum

528

undulatum L. J. Ethnopharmacol. 2003, 84, 5-9.

529

42. Boelsterli, U. A.; Zimmerman, H. J.; Kretz-Rommel, A., Idiosyncratic liver toxicity

530

of nonsteroidal antiinflammatory drugs: molecular mechanisms and pathology. Crit.

531

Rev. Toxicol. 1995, 25, 207-235.

532

43. Pumford, N. R.; Halmes, N. C., Protein targets of xenobiotic reactive

533

intermediates. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 91-117.

534

44. Pirmohamed, M.; Madden, S.; Park, B. K., Idiosyncratic drug reactions. Metabolic

535

bioactivation as a pathogenic mechanism. Clin. Pharmacokinet. 1996, 31, 215-230.

536

45. Park, B. K.; Coleman, J. W.; Kitteringham, N. R., Drug disposition and drug

537

hypersensitivity. Biochem. Pharmacol. 1987, 36, 581-590.

538

46. Pohl, L. R.; Satoh, H.; Christ, D. D.; Kenna, J. G., The immunologic and metabolic

539

basis of drug hypersensitivities. Annu. Rev. Pharmacol. Toxicol. 1988, 28, 367-387.

540

47. Meech, R.; Miners, J.O.; Lewis, B.C.; Mackenzie, P.I., The glycosidation of

541

xenobiotics and endogenous compounds: versatility and redundancy in the UDP

542

glycosyltransferase superfamily. Pharmacol. Ther. 2012, 134, 200–218.

543

48. Achour, B.; Rostami-Hodjedan, A.; Barber, J., Protein expression of various hepatic

544

uridine 5’-diphosphate glucuronosyltransferase (UGT) enzymes and their correlations:

545

A meta-analysis. Biopharm. Drug Dispens. 2014, 35, 353–361.

546

49. Boelsterli U.A., Acyl glucuronides: mechanistic role in drug toxicity. Curr. Drug

547

Metab. 2011, 12, 213-214.

548

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FIGURE CAPTIONS

550

Figure 1. Chemical structures of rhein, RG3 and hyperoside (A) and synthetic method

551

of RG3 (B).

552

Figure 2. MRM chromatograms of incubated samples of rhein with human liver

553

microsomes for 40 min (A), formation rates of glucuronides of rhein by human, rat,

554

mouse, monkey or dog liver microsomes (B) and typical MS1 and MS2 spectra of

555

rhein glucuronides (C). GlcA: glucuronic acid.

556

Figure 3. Stability of RG1, RG2 and RG3 in incubation solution (A), MRM

557

chromatograms incubation of RG3in PBS for 5 h (B) and stability of RG3 in PBS (C).

558

Figure 4. MRM chromatograms incubation of RG3 with GSH for 5 h (A), time course

559

of RG3 in the incubation with GSH (B) and MS spectra of RGSH (C). pGlu:

560

pyroglutamic acid.

561

Figure 5. Formation rates of RG1 and RG2 (A) and RG3 (B) by incubation of rhein

562

with recombinant UGTs for 40 min.

563

Figure 6. Effects of rhein concentration on formation of RG1, RG2 and RG3 by

564

different recombinant human UGTs.

565

Figure 7. Effects of different concentration of rhein and RG3 on the viability of

566

HepG2 cells. *** p