In Vitro and in Vivo Studies of the Electrophilicity of Physcion and its Oxidative Metabolites

Article abstract of DOI:10.1021/acs.chemrestox.8b00026

Physcion (1,8-dihydroxy-3-methoxy-6-methyl-9,10-anthracenedione) is a bioactive component found in Polygoni Multiflori Radix (PMR), which has been widely used as traditional Chinese medicine. Unfortunately, studies showed hepatotoxicity of PMR during its clinical use. The mechanisms of its toxic action remain unknown. The major objectives of this study were to characterize oxidative metabolites of physcion in vitro and in vivo and to determine the electrophilicity of the parent compound and its oxidative metabolites. Five oxidative metabolites (M1-M5) were detected in rat liver microsomal incubations after exposure to physcion, and the formation of the metabolites was NADPH dependent. M1-M4 were monohydroxylation metabolites, and M5 was O-demethylation metabolite. A total of three N-acetylcysteine (NAC) conjugates (M6-M8) were observed in rat liver microsomes fortified with NAC as a trapping agent. M6 was derived from M4 conjugated with a molecule of NAC; M7 and M8 originated from parent compound physcion adducted with a molecule of NAC, respectively. M1-M8 were also observed in urine of rats given physcion. HLM incubations produced four oxidative metabolites and two NAC conjugates. The structures of M3, M7, and M8 were characterized by LC-Q-TOF MS and NMR. Recombinant P450 enzyme incubations demonstrated that CYPs2C19, 1A2, 2B6, and 3A4 were mainly involved in hydroxylation of physcion. The metabolism study assisted us to better understand the mechanisms of physcion-induced hepatotoxicity.

Full text of DOI:10.1021/acs.chemrestox.8b00026

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In Vitro and in Vivo Studies of the Electrophilicity
of Physcion and its Oxidative Metabolites
Xiaotong Qin, Ying Peng, and Jiang Zheng
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00026 • Publication Date (Web): 18 Apr 2018
Downloaded from http://pubs.acs.org on April 19, 2018
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In Vitro and in Vivo Studies of the Electrophilicity of Physcion and its Oxidative
Metabolites
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Xiaotong Qin,† Ying Peng,†* and Jiang Zheng†§*
†
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P.R.
China
§
State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of
Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550025, P.R.
China
Corresponding Authors:
Jiang Zheng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua
Road, Shenyang, Liaoning, 110016, P. R. China
State Key Laboratory of Functions and Applications of Medicinal Plants, Key
Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, 9
Beijing Road, Guiyang, Guizhou, 550025, P.R. China
Email: zhengneu@yahoo.com
Tel: +86ꢀ24ꢀ23986361; Fax: +86ꢀ24ꢀ23986510
Ying Peng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua
Road, Shenyang, Liaoning, 110016, P. R. China
Email: yingpeng1999@163.com
Tel: +86ꢀ24ꢀ23986361; Fax: +86ꢀ24ꢀ23986510
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Abstract
Physcion (1,8ꢀdihydroxyꢀ3ꢀmethoxyꢀ6ꢀmethylꢀ9,10ꢀanthracenedione) is a
bioactive component found in Polygoni Multiflori Radix (PMR) which has been
widely used as traditional Chinese medicine. Unfortunately, studies showed
hepatotoxicity of PMR during its clinical use. The mechanisms of its toxic action
remain unknown. The major objectives of this study were to characterize oxidative
metabolites of physcion in vitro and in vivo and to determine the electrophilicity of
the parent compound and its oxidative metabolites. Five oxidative metabolites (M1–
M5) were detected in rat liver microsomal incubations after exposure to physcion, and
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the formation of the metabolites was NADPH dependent.
monoꢀhydroxylation metabolites, and M5 was ꢀdemethylation metabolite. A total
of three ꢀacetylcysteine (NAC) conjugates (M6–M8) were observed in rat liver
M1–M4 were
O
N
microsomes fortified with NAC as a trapping agent. M6 was derived from M4
conjugated with a molecule of NAC; M7 and M8 originated from parent compound
physcion adducted with a molecule of NAC, respectively. M1–M8 were also
observed in urine of rats given physcion. HLM incubations produced four oxidative
metabolites and two NAC conjugates. The structures of M3, M7 and M8 were
characterized by LCꢀQꢀTOF MS and NMR. Recombinant P450 enzyme incubations
demonstrated that CYPs2C19, 1A2, 2B6, and 3A4 were mainly involved in
hydroxylation of physcion. The metabolism study assisted us to better understand
the mechanisms of physcionꢀinduced hepatotoxicity.
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Introduction
Polygonum multiflorum Thunb., known as Heshouwu in China and Foꢀti in
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North America, is an important Chinese herbal medicine and is officially listed in the
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Chinese Pharmacopoeia. Pharmacological studies and clinical practice demonstrated
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that it possesses antiꢀaging, antiꢀoxidative, antiꢀinflammatory, and antiꢀcancer effects.
In addition, it also exhibits effective outcome in the treatment of premature greying of
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hair, lumbago, spermatorrhea, leucorrhea and constipation.
Meanwhile,
phytochemical studies shown that anthraquinones (AQs) are one of main bioactive
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components in Polygonum multiflorum Thunb.. In addition to their antiꢀbacterial
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properties, AQs exert antiꢀfungal and antiꢀcancer effects and are considered to be
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useful for the disintegration and elimination of urinary stones. However, with
chronically used of Polygoni Multiflori Radix (PMR), the roots of Polygonum
multiflorum Thunb., hepatic adverse effect were constantly reported since 1990s in
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China and other countries.
Acute hepatitis was the most frequently reported
Since many AQꢀcontaining herbal medicines have been
reported to induce hepatic damages, AQs are assumed to be responsible for the
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hepatic adverse effect.
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observed hepatotoxicity.
Physcion (1,8ꢀdihydroxyꢀ3ꢀmethoxyꢀ6ꢀmethylꢀ9,10ꢀanthracenedione), an AQ
derivative, has been isolated and characterized from both terrestrial and marine
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sources.
In particular, physcion is rich in PMR which is widely used as
traditional Chinese medicine. Physcion reportedly possessed the efficacies of
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antiꢀinflammatory, antiꢀmicrobial, and antineuroectodermal tumor properties.
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Physcion was found to be cytotoxic in HeLa, A549, HLꢀ60, and SW680 cells.
To our best knowledge, the causal link between physcion and its hepatic
lesions, along with exact metabolic pathway of physcion, remains uncertain. The
present study was undertaken to investigate (1) biotransformation of physcion in vitro
and in vivo and (2) the electrophilicity of physcion and its metabolites possibly related
with the observed cytotoxicity.
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EXPERIMENTAL PROCEDURES
Chemicals and Materials. Physcion and emodin with purity of >98% were
supplied by Chengdu Pufeide Biologic Technology Co. Ltd. (Chengdu, China).
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N
ꢀAcetylcysteine (NAC), nicotinamide adenine dinucleotide phosphate reduced
(NADPH) and propranolol were obtained from SigmaꢀAldrich (St. Louis, MO).
Human liver microsomes (HLMs) and recombinant human P450 enzymes were
purchased from BD Gentest (Woburn, MA). Rat (Sprague−Dawley, male) liver
microsomes (RLMs) were prepared in our laboratory, according to a previously
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published method . All organic solvents were acquired from Fisher Scientific
(Springfield, NJ). Distilled water was purchased from Wahaha Co. Ltd. (Hangzhou,
China). All of the reagents and solvents were analytical or highꢀperformance liquid
chromatography grade.
Microsomal Incubations. A stock solution of physcion was prepared in dimethyl
sulfoxide (DMSO). The incubation mixtures were prepared in a final volume of 250
ꢀL, containing 1.0 mg/mL RLM or HLM protein, 3.2 mM MgCl
2
, 20 mM NAC, and
25 ꢀM physcion. The microsomal reactions were initiated by adding NADPH (1.0
mM) and conducted at 37 °C in a gentle shaking water bath for 30 min. The
negative control without NADPH was included to ensure that the metabolism of
physcion was NADPH dependent. The reactions were quenched with an equal
volume of iceꢀcold acetonitrile, followed by vortexꢀmixing and centrifuging at 19,000
g
for 10 min to remove precipitated protein. A 5 ꢀL aliquot of supernatants was
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injected into an LC–MS/MS system for analysis. All incubations were carried out in
duplicate.
Chemical Synthesis of Physcion Metabolites. The methyl moiety of physcion was
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oxidized to prepare M1, based on a reported procedure.
was dissolved in acetic acid (1 mL), followed by addition of acetic anhydride (1.8 mL)
and concentrated sulfuric acid (18 L) dropwise. The resulting mixture was stirred
at 70 °C for 30 min and poured onto ice water to afford crystals of
,3ꢀdiacetylphyscion. With the crude products (25 mg) dissolved in a mixture of
glacial acetic acid (1 mL) and acetic anhydride (1 mL), an acetic acid solution (5 mL)
containing chromium (VI) oxide (CrO , 600 mg) was dropwise added. The mixtures
were stirred for 24 h at room temperature, whereafter, blown dried under a nitrogen
stream and reꢀdissolved in dry tetrahydrofuran (THF) (500 L).
Boraneꢀmethylsulfide complex (175 L) was added to the THF precooled at 0 °C.
Briefly, physcion (40 mg)
ꢀ
1
3
µ
ꢀ
The reaction mixtures were further stirred at 0 °C for 1 h, and the reaction was
terminated by mixing with water (1 mL). The resulting mixture was extracted with
3
ethyl acetate, and the extracts were washed with 5% NaHCO aqueous solution,
followed by addition of 1 mL of potassium hydroxide solution (0.2 M). The mixture
was stirred at 75 °C for 15 min. The resultant mixture was acidified and extracted
with ethyl acetate, and the organic solvent was evaporated to acquire M1.
M2 was prepared by hydroxylation of physcion using boric acid, according to a
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reported procedure.
Briefly, physcion (10 mg) was mixed with 1 mL of 65%
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oleum including boric acid (50 mg) in a sealed flask. The mixture was stirred at
room temperature for 6 h and then was allowed to stand for 3 days. The solution
was poured cautiously onto ice (25 g), followed by boiling for 10 min and chilling to
room temperature. The resulting orange precipitates were harvested and then
dissolved in 20 mL of 0.2 M potassium hydroxide solution under nitrogen. The
mixture was heated on a boiling water bath for 15 min and the acidified with 5 mL of
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5% HCl solution. The acidic solution was extracted with ethyl acetate (5 mL×3),
and the organic phase was washed with H O (5 mL×3) and concentrated by
2
evaporating under vacuum. The residue was recrystallized from dilute acetic acid
and submitted to the LCꢀMS/MS analysis.
M3 and M4 were obtained from hydroxylation of physcion by a published
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protocol.
In brief, physcion (40 mg) and potassium persulfate (104 mg) were
dissolved in sulfuric acid (2 mL). After being shaken for 10 min at room
temperature, 104 mg potassium persulfate was added with stirring. After 45 min, the
reaction was terminated by addition of 40 mL of water and 400 mg sodium
metabisulfite. The mixture was extracted with butanol (6 mL) and washed with 5%
HCl (20 mL) then water (20 mL). The solvent was evaporated and the residue was
subjected to the LCꢀMS/MS system for analysis. Finally, M3 was purified by a
semiꢀpreparative HPLC system and characterized by mass spectrometry and NMR.
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HꢀNMR (DMSOꢀd , 600 MHz): δ7.11 (1H, s, Hꢀ7), 7.34 (1H, s, Hꢀ4), 7.49 (1H, d,
J
=1.58 Hz, Hꢀ5).
NAC conjugates M7 and M8 were synthesized by direct reaction of NAC with
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physcion. Physcion and NAC were dissolved in potassium phosphate buffer (pH =
7.4) and stirred at 37 °C for 2 h. The resultant mixture was concentrated by rotary
evaporation and reconstituted with the initial mobile solvent and submitted to
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LCꢀMS/MS analysis. M7 and M8 were purified by a semiꢀpreparative HPLC system
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and characterized by mass spectrometry and NMR. M7: H NMR (DMSOꢀ
d
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, 600
MHz): δ2.42 (3H, s, Hꢀ11), 4.01 (3H, s, Hꢀ12), 7.19 (1H, s, Hꢀ7), 7.27 (1H, s, Hꢀ4),
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7
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.52(1H, s, Hꢀ5); C NMR (DMSOꢀd , 600 MHz): δ21.50 (Cꢀ11), 56.81 (Cꢀ12),
02.10 (Cꢀ4), 110.60 (Cꢀ9a), 113.60 (Cꢀ8a), 116.60 (Cꢀ2), 120.40 (Cꢀ5), 124.40 (Cꢀ7),
32.70 (Cꢀ4a), 133.80 (Cꢀ10a), 148.60 (Cꢀ6), 159.60 (Cꢀ1), 161.60 (Cꢀ8), 165.40
1
(
Cꢀ3), 181.50 (Cꢀ10) 189.90 (Cꢀ9); M8: H NMR (DMSOꢀ
d
6
, 600 MHz): δ6.87 (1H, d,
J
=2.26 Hz, Hꢀ2), 7.18 (1H, d,
J=2.33 Hz Hꢀ4), 7.53 (1H, s, Hꢀ5).
Synthetic products were purified on a YMCꢀPack ODSꢀA column (250×10 mm,
Sꢀ5, 12 nm) (YMC Co., Ltd, Japan), and the purification was achieved on a
semiꢀpreparative HPLC system. The rate was set 2.5 mL/min.
Animal Experiments. All animal manipulations met the requirements approved by
the Ethics Review Committee for Animal Experimentation of Shenyang
Pharmaceutical University (Shenyang, China). Male SpragueꢀDawley rats (200±20
g) were obtained from the Animal Center of Shenyang Pharmaceutical University and
were kept in a controlled environment (temperature of 25 °C and 12 h dark/light cycle)
with maintaining on standard metabolism cages. Blank urine samples carefully
conserved on ice were collected after a 12ꢀh overnight with free access to water prior
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to the treatment. Physcion dissolved in corn oil (13.6 mg/mL) were administered
intraperitoneally at 75 mg/kg. Urine samples carefully preserved on ice were
collected during the time periods of 0–12, 12–24, and 24–48 h after the procedure.
During the experiments, the rats were allowed unlimited access to food and water.
The collected urine samples were stored at ꢀ80 °C until analysis.
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Sample Preparation for LC–MS/MS Analysis. Triple volumes of acetonitrile
were added to the urine samples and the mixtures were vortexed for 3 min and
centrifuged at 19,000
concentrated under a nitrogen flow at 40 °C. The resulting concentrates were
reconstituted with 10% acetonitrile in water (100 L) and centrifuged at 19,000 for
L aliquot of the supernatants was injected into the LCꢀMS/MS for
g for 10 min. The supernatants were collected and
µ
g
10 min. A 3
analysis.
ꢀ
Recombinant Human P450 Enzyme Incubations. Physcion was individually
incubated with nine recombinant human P450 enzyme (100 nM), including P450s
1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. Other conditions were
equivalent to the microsomal incubation experiments. An equal volume of iceꢀcold
acetonitrile which contained propranolol (40 ng/mL) as internal standard was mixed
with incubation fluid to terminate the reactions. After vortexed for 2 min, the
resulting samples were centrifuged at 16,000
g for 10 min. The supernatants (5 ꢀL)
were injected into the LCꢀMS/MS for analysis. Each incubation was carried out in
duplicate. A total normalized rate method was applied. The rates of metabolite
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formation in individual incubations with recombinant P450 enzymes were multiplied
by the mean specific content of the corresponding P450 enzyme in HLMs to obtain
33
the normalized reaction rates of each enzyme.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
LC-MS/MS Method. All samples were analyzed on an AB SCIEX Instruments
5500 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA,
USA) interfaced online with a 1260 infinity system (Agilent Technologies, Santa
Clara, CA). The metabolite separation was achieved on an Accuore C18 column (4.6
×
150 mm, 5 ꢀm; Waters, Ireland). Gradient elution was performed with acetonitrile
containing 0.1% formic acid (solvent A) and 0.1% formic acid in water (solvent B) as
follow: 10 % A at 0−2 min; 10−95 % A at 2−12 min; 95−95 % A at 12−17 min;
95−10 % A at 17−18 min; and 10−10 % A at 18−20 min. The flow rate was kept at
0.8 mL/min.
The samples were analyzed in positiveꢀion mode with
multipleꢀreaction monitoring (MRM) scanning. After tuning, the optimized MS
instrument parameters were set at the following values: ion spray voltage (IS),
entrance potential (EP) and ion source temperature were 5,500 V, 10 V, and 650 °C
respectively; ion source gas 1, 50 psi; ion source gas 2, 50 psi; curtain gas, 35 psi.
To achieve the highest sensitivity possible, MRM was used to selectively monitor
with ion transitions (corresponding to declustering potential DP, collision energy CE)
m/z 301→199 (100, 45) for hydroxylation metabolites, m/z 271→197 (100, 45) for
O
ꢀdemethylation metabolite, m/z 446→315 (100, 40) and 462→333 (100, 20) for
NAC conjugates, m/z 260→116 (77, 25) for propranol (internal standard), and m/z
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
85→168(77, 25) for physcion.
In addition, a 1260 infinity LC system (Agilent Technologies, Boblingen,
Germany) with an AB SCIEX 4000 QꢀTrap MS (Applied Biosystems, Foster City, CA)
were applied to trigger the enhanced product ion (EPI) scans by analyzing MRM
signals which intended to gain abundant fragmentation information for target
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
compounds.
By using informationꢀdependent acquisition (IDA) to trigger
acquisition of EPI spectra for ions exceeding 1000 cps with exclusion of former target
ions after three occurrences for 10 s. EPI scan was run in positive mode at a scan
range of product ions from m/z 50 to 310 or 50 to 500. All data were processed
using the AB SCIEX Analyst 1.6.2 software (Applied Biosystems).
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RESULTS
Mass Spectrometric Behavior of Physcion. To facilitate the characterization of
physcion metabolites, the chromatographic and mass spectrometric properties of
parent compound were determined as an initial step. Positive mode was employed
throughout the study, because of the higher sensitivity, ionization efficiency and more
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fragment ions obtained.
Physcion showed fragment ions of m/z 239
+
+
+
(
(
(
[M+HꢀH
2
2
OꢀCO] ), 211 ([M+HꢀH
2
Oꢀ2CO] ), 183 ([M+HꢀH
2
Oꢀ3CO] ), 252
+
+
+
[M+HꢀH
OꢀCH
3
] ), 242 ([M+HꢀCOꢀCH
3
] ), 196 ([M+HꢀH
2
Oꢀ2COꢀCH
3
] ), 168
+
+
+
[M+HꢀH Oꢀ3COꢀCH ] ), and 140 ([M+HꢀH Oꢀ4COꢀCH ] ), along with [M+H] as
2
3
2
3
m/z 285 (Fig. 1), which results from the loss of CH
2 2 2 2 3 3 2 4 5
O , C H O , C H O , CH O,
C
2
H
3
O, C and C respectively.
3
H
5
O
3
C
4
H
5
O
4
5 5 5
H O
In Vitro Oxidation of Physcion. Physcion was incubated in RLMs fortified with
NADPH and NAC, and five oxidative metabolites (M1–M5) (Fig. 2B, 5B) and three
NAC conjugates (M6–M8) were detected (Fig. 6B, 7B). Noteworthy, M7 and M8
were found in RLM incubations with or without NADPH (Fig. 7A, 7B), while no
M1ꢀM6 were observed when NADPH was absent in microsomal incubations (Fig. 2A,
5A, 6A). This indicates that the information of M1ꢀM6 was NADPHꢀdependent.
Metabolites M1ꢀM4 were oxidative metabolites of physcion. Formation of
M1ꢀM4 was monitored by acquiring precursor/product ion pair m/z 301/199 in
positive mode. M1ꢀM4 eluted out at retention time of 11.0, 12.3, 12.7, and 13.5 min,
respectively (Fig. 2B). With MRM–EPI scanning (ion transition m/z 301/199), the
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MS/MS spectra of M1ꢀM4 were gained, which showed characteristic fragment ions of
+
+
m/z
[MHꢀH
184
([MHꢀH
2
Oꢀ3COꢀCH
3
] ),
199
([MHꢀH
2
Oꢀ3CO] ),
212
+
+
+
(
2
Oꢀ2COꢀCH
3
] ), 227 ([MHꢀH
2
Oꢀ2CO] ), and 255 ([MHꢀH
2
OꢀCO] ) (Fig. 3C,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3D, 4B, 4C). It is worth mentioning that the product ions of m/z 184, 199, 212, 227,
and 255 were 16 Da more than the corresponding ions at m/z 168, 183, 196, 211, and
2
39 found in mass spectrum of the parent compound, indicating that M1ꢀM4 were
monoꢀhydroxylation metabolites of physcion. M1ꢀM4 shared the same molecular
ion at 301, along with similar fragment patter. This indicates that the four
m/z
metabolites were isomers.
M5, which eluted out at 13.4 min, was characterized by monitoring
precursor/product ion pair m/z 271/197 (Fig. 5B). The value of its protonated
molecule (m/z 271) was 14 Da lower than that of physcion. The fragment ions at m/z
169, 197, and 225 were derived from parent fragmentation of
m/z 183
+
+
+
(
[MHꢀH Oꢀ3CO] ), 211 ([MHꢀH Oꢀ2CO] ), 239 ([MHꢀH OꢀCO] ), respectively (Fig.
2
2
2
5F). This suggests that M5 was the
O
ꢀdemethylation metabolite of physcion.
The formation of M6 was monitored by acquiring ion pair of m/z 462→333 (Fig.
6B). The MS/MS spectrum of M6 (Rt = 10.7 min) demonstrated the major fragment
ions associated with cleavage of the NAC moiety (Fig. 6F). The product ion at m/z
30 corresponded to NAC moiety without the sulfur, and the fragment ion at m/z 162
1
was derived from NAC moiety with the sulfur. The product ion at m/z 333 was
associated with the characteristic neutral loss of 129 Da from NAC conjugate M6.
Since the molecular weight of NAC is 163 Da, the observed molecular ion of m/z 462
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suggests that M6 resulted from a hydroxylation metabolite of physcion conjugated
with a molecule of NAC.
M7 and M8 eluted out at 11.8 and 12.1 min respectively, showing their
molecular ions at m/z 446 (Fig. 7B). The MS/MS spectra of M7 and M8 were
acquired by MRMꢀEPI scanning (ion transition m/z 446/315) which displayed
characteristic fragment ions of m/z 130, 162, 315, and 317 (Fig. 8B, 8C). Similarly,
the product ion at m/z 130 corresponded to NAC moiety without the sulfur, and the
fragment ion at m/z 162 was derived from NAC moiety with the sulfur. In addition,
the product ion at m/z 317 matched the characteristic neutral loss of 129 Da for the
NAC conjugates (M7 and M8). Therefore, M7 and M8 were isomers which shared
the same molecular ions at m/z 446, possibly resulting from the parent compound
conjugated with a molecule of NAC at two different positions.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Interestingly, we failed to detect M3 and M6 in HLM incubations which were
conducted under similar condition as that of RLM incubations (Fig. 9).
Chemical Synthesis of Metabolites of Physcion
out to verify the structure of M1–M8 obtained from the microsomal incubations above.
Physcion oxidized in series of chemical reactions with CrO and
.
Chemical synthesis was carried
a
3
boraneꢀmethylsulfide complex to acquire a product which revealed the same
chromatographic and mass spectral behaviors as that of M1 formed in RLM
incubations (Fig. 3A, S1A). Since the oxidation protocol was reported to selectively
31
hydroxylate the benzylic carbon, we proposed that M1 was 6ꢀhydroxyphyscion.
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Similarly, hydroxylation of physcion in oleum and boric acid also got an outcome in
which the product had the same chromatographic and mass spectral behaviors as that
of M2 produced in the RLM incubations (Fig. 3B, S1C). Besides, treatment of
physcion with persulfate in sulfuric acid generated two products which had the same
chromatographic and mass spectral behaviors as that of M3 and M4 generated in the
RLM incubations (Fig. 4A, S2A, S2C). M3 was purified and characterized by
LCꢀQꢀTOF MS analysis (Table 1) and NMR. Three aromatic proton resonances
were observed at 7.11, 7.34, and 7.49 ppm responsible for the protons at Cꢀ7, Cꢀ4 and
Cꢀ5. However, no proton resonance was found to be responsible for the proton at
Cꢀ2. This made us conclude that hydroxylation occurred at Cꢀ2. Together, the
synthetic work allowed us to assign M3 as 2ꢀhydroxyphyscion. The LCꢀQꢀTOF MS
data of M4 (Table 1) revealed the molecular ion which match the molecular formula
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(C16
H
12
O
6
) responsible for hydroxylated physcion.
The structure of M5 was verified by comparing its chromatographic and mass
spectral properties with that of authentic standard emodin. As expected, M5 was
identified as emodin (Fig. 5C, 5H).
In order to determine the source of M6, synthetic M1, M2, M3 and M4 were
individually incubated with NAC in RLMs. M4ꢀNAC reaction generated a product
sharing the same chromatographic and mass spectral identities as those of M6
produced in microsomal incubations (Fig. 6C, 6H). Hence, M6 originated from M4
conjugated with a molecule of NAC.
Direct mixing of physcion with NAC in PBS buffer generated two products
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which revealed the same chromatographic and mass spectral behaviors as those of M7
and M8 produced in the RLM incubations (Fig. 8A, S3A, S3C). M7 and M8 were
then purified and characterized by LCꢀQꢀTOF MS (Table 1) and NMR. Figure S4
shows the HMBC (longꢀrange protonꢀcarbon heteronuclear multiple bond correlation)
spectrum of M7. Three aromatic proton resonances at 7.19, 7.27, and 7.52 ppm are
responsible for the protons at Cꢀ7, Cꢀ4 and Cꢀ5. Carbon signals at δC21.50, 110.60,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
113.60, 120.40, 124.40 and 181.5 ppm corresponded to Cꢀ11, Cꢀ9a, Cꢀ8a, Cꢀ5, Cꢀ7
and Cꢀ10, respectively. Additionally, the HMBC spectrum revealed that Cꢀ9a and
Cꢀ10 had correlations with the proton at 7.27 ppm; Cꢀ8a, Cꢀ7, and Cꢀ10 had
correlations with the proton at 7.52 ppm; Cꢀ11, Cꢀ5, and Cꢀ8a had correlations with
the proton at 7.19 ppm. The NMR spectrum suggests that NAC is attached to Cꢀ2 of
physcion.
1
The HꢀNMR spectrum of M8 showed three aromatic proton resonances at 6.87,
7.18, and 7.53 ppm responsible for the protons at Cꢀ2, Cꢀ4 and Cꢀ5. Apparently, no
proton resonance responded to the proton at Cꢀ7. This made us conclude that M8
was NAC conjugation took place at Cꢀ7 of physcion.
Metabolism of Physcion in Rats. In vivo metabolism of physcion was investigated
by analyzing urine samples of rats with or without treatment of physcion. M1ꢀM8
were found in the urine of the animals treated with physcion, and no such metabolites
were observed in the control urine samples (Fig. 2C, 2D, 5D, 5E, 6D, 6E, 7C, 7D).
The chromatographic and mass spectral behaviors of the metabolites detected in the
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
urine were in agreement with those produced in microsomal incubations (Fig. 2D,
S1B, S1D, S2B, S2D, 5E, 5G, 6E, 6G, 7D, S3B, S3D)
.
P450 Enzymes Involved in Physcion Biotransformation. Recombinant human
P450s were used to determine which P450 enzymes were responsible for the
metabolism of physcion. Nine human recombinant P450 enzymes were individually
incubated with physcion and NAC, and the formation of M1ꢀM2, and M4ꢀM5 was
monitored to determine the specific P450 enzymes involved in the biotransformation
of physcion (Fig. 10). P450 1A2 was found to be the major enzyme contributing to
the production of M1. P450 2C19 was the primary enzyme participating in the
generation of M2. The formation of M4 was catalyzed by P450 2C19 and then 3A4.
P450 2B6 was the major enzyme catalyzing the formation of M5, followed by P450s
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1A2 and 2C19. However, M3 and M6 were not found in recombinant human P450
incubations.
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DISCUSSION
Physcion is a bioactive component in PMR which has been widely consumed
in China and North America. However, studies also showed multiple adverse effects
during its clinical trials, such as hepatotoxicity of PMR, but the mechanisms of the
toxicities remain unknown. Investigation of metabolism pathways of physcion
would be helpful for the understanding of the mechanisms of physcion toxic action.
Firstly, we studied the mass spectrometric behavior and fragmentation pattern of
physcion (Fig. 1), which would help the structural characterization of physcion
metabolites. Whereafter, RLM incubations were conducted with physcion, using
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NAC as the capture reagent. Comparison of the changes in molecular masses (ꢁM
)
2
and MS spectral patterns of metabolites with that of parent drug could assist us to
better characterize the metabolites. Four hydroxylation metabolites (M1−M4) (Fig.
2B), one
M6–M8) were detected by LCꢀMS/MS (Fig. 6B, 7B). Apparently, M1 was found to
Oꢀdemethylation metabolite (M5) (Fig. 5B), and three NAC conjugates
(
be the primary metabolite among the four hydroxylation metabolites (M1−M4) (Fig.
S5), based on their relative abundance obtained from LCꢀMS/MS data. Interestingly,
the metabolism of physcion showed some species difference. No M3, along with
significantly decreased formation of M2/M4, was detected in the HLM incubations
(Fig. 9) conducted under similar condition as RLM incubations. Besides, no M6 was
found in the HLM incubations, possibly resulting from the decreased generation of
M2/M4.
Chemical synthesis was carried out to verify the metabolites. M1 was
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
synthesized by hydroxylation of the benzylic carbon of physcion using CrO and
boraneꢀmethylsulfide complex (Fig. 3A). M2 was prepared by hydroxylation of
physcion in oleum and boric acid (Fig. 3B). M3 and M4 were obtained from
treatment of physcion with persulfate in sulfuric acid (Fig. 4A). The NMR spectrum
demonstrated that M3 was 2ꢀhydroxyphyscion. Unfortunately, we failed to obtain
the NMR data of M1, M2 and M4, due to the lower yield and instability of the
products. M5 was identified as emodin using the authentic compound (Fig. 5C, 5H).
The mass spectrum of M6 suggests the metabolite was a NAC conjugate derived from
hydroxylation metabolites of physcion. The origin of M6 was determined by
incubation of individual synthetic M1, M2, M3 and M4 in RLM fortified with NAC.
The result demonstrated M6 was derived from M4 conjugated with a molecule of
NAC (Fig. 6C, 6H).
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M7 and M8 were isomers resulting from direct reaction of physcion with NAC.
We detected M7 and M8 in both RLM and HLM incubations with or without NADPH
(Fig. 7A, 7B, 9E, 9F). This suggests that the formation of M7 and M8 was
independent on P450 enzymes. The two metabolites were generated by direct
adduction, followed by autoꢀoxidation possibly mediated by air. Additionally, the
presence of NADPH was found to attenuate the formation of the two conjugates (Fig.
7A, 7B, 9E, 9F). This may explain that NADPH initiated P450ꢀmediated oxidative
metabolism of physcion and made physcion less available for the formation of M7
and M8. The NMR data showed that M7 was the conjugate with an NAC attached
to Cꢀ2 of physcion (Fig. S4), while M8 was the adduct with an NAC conjugated at
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Cꢀ7.
Metabolism of physcion in vivo was investigated by analyzing the urine
samples of rats treated with physcion. As expected, M1ꢀM8 were detected in the
urine, and the urinary metabolites illustrated similar retention time and mass spectral
fragment patterns with those of the corresponding metabolites produced in RLM
incubations (Fig.2D, S1B, S1D, S2B, S2D, 5E, 5G, 6E, 6G, 7D, S3B, S3D).
Proposed metabolic pathways of physcion are displayed in Scheme 1.
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ꢀdemethylation of physcion generated M5 which is emodin, a major AQ derivative
found in PMR. Our previous work demonstrated that emodin was chemically
34
reactive to thiolꢀcontaining agents.
Studies showed that emodin could cause a
35
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37
variety of toxic effects such as nephrotoxicity, genotoxicity and hepatotoxicity.
Hence, emodin, as metabolite of physcion, might contribute to physcionꢀinduced
hepatocyte toxicity.
In conclusion, five oxidative metabolites (M1−M5) of physcion and three
related NAC conjugates (M6–M8) were detected in RLM incubations and in urine
samples of rats after exposure to physcion, while HLM incubations produced four
oxidative metabolites and two NAC conjugates. P450 1A2, 2C19 and 2B6 were the
primary enzymes mediating the hydroxylation of physcion. The parent compound
and all oxidative metabolites showed electrophilicity. The metabolism work would
allow us to better understand the molecular mechanisms of physcionꢀinduced
hepatotoxicity.
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Author Contributions
The manuscript was written by Jiang Zheng and Xiaotong Qin. The research design
was performed by Jiang Zheng and Ying Peng. Experiments were performed and
data analysis by Xiaotong Qin.
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Funding sources
This work was supported in part by the National Natural Science Foundation of China
(No. 81430086, 81373471 and 81773813).
Supporting Information Available
MS/MS spectra of M1ꢀM4 and M7ꢀM8 generated in chemical synthesis and found in
urine samples of rats treated with physcion were supplied as supporting information.
HMBC (longꢀrange protonꢀcarbon heteronuclear multiple bond correlation) spectrum
of M7 was also provided, along with the relative abundance of metabolites (M1ꢀM8)
obtained from LCꢀMS/MS analysis of RLM incubations containing physcion in the
presence of NADPH.
http://pubs.acs.org.
Notes
This material can be accessed via the Internet at
The authors declare no competing financial interest.
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Abbreviations: CE, collision energy; DMSO, dimethyl sulfoxide; DP, declustering
potential; EPI, enhanced product ion; HMBC, longꢀrange protoncarbon heteronuclear
multiple bond correlation; IDA, informationꢀdependent acquisition; LCꢀMS/MS,
liquid chromatographyꢀtandem mass spectrometry; MRM, multipleꢀreaction
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monitoring; NAC,
Nꢀacetyl cysteine; NADPH, βꢀnicotinamide adenine dinucleotide
2′ꢀphosphate reduced tetrasodium salt; NMR, nuclear magnetic resonance; PMR,
Polygoni Multiflori Radix; THF, tetrahydrofuran.
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Figure Legends
Figure 1. MS/MS spectrum of physcion.
Figure 2. Chromatograms of M1, M2, M3 and M4. Extracted ion (m/z 301/199
for M1, M2, M3 and M4) chromatograms obtained from LCꢀMS/MS analysis of RLM
incubations containing physcion in the absence (A) or presence (B) of NADPH.
Extracted ion (m/z 301/199 for M1, M2, M3 and M4) chromatograms obtained from
LCꢀMS/MS analysis of urine samples of rats treated with vehicle (C) or physcion (D).
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Figure 3. Characterization of M1 and M2. Extracted ion (m/z 301/199)
chromatograms obtained from LCꢀMS/MS analysis of synthetic M1 (A) and synthetic
M2 (B). MS/MS spectra of M1 generated in RLM incubations (C) and MS/MS
spectra of M2 generated in RLM incubations (D).
Figure 4. Characterization of M3 and M4. A: Extracted ion (m/z 301/199)
chromatogram obtained from LCꢀMS/MS analysis of synthetic M3 and M4. MS/MS
spectra of M3 generated in RLM incubations (B) and MS/MS spectra of M4
generated in RLM incubations (C).
Figure 5. Characterization of M5. Extracted ion (m/z 271/197) chromatograms
obtained from LCꢀMS/MS analysis of RLM incubations containing physcion in the
absence (A) or presence (B) of NADPH. (C) Extracted ion (m/z 271/199)
chromatogram obtained from LCꢀMS/MS analysis of chemistry reference substance
of emodin. Extracted ion (m/z 271→197, M5) chromatograms obtained from
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