Metabolic mechanism and anti-inflammation effects of sinomenine and its major metabolites N-demethylsinomenine and sinomenine-N-oxide

Article abstract of DOI:10.1016/j.lfs.2020.118433

Aims: Sinomenine (SIN) is clinically used as an anti-rheumatic drug. However, the metabolic and pharmacological mechanisms of SIN combined with its metabolites are unclear. This study aims to explore the cyclic metabolic mechanism of SIN, the anti-inflammation effects of SIN and its major metabolites (N-demethylsinomenine (DS) and sinomenine-N-oxide (SNO)), and the oxidation property of SNO. Materials and methods: SIN was administrated to rats via gavage. Qishe pills (a SIN-containing drug) were orally administrated to humans. The bio-samples were collected to identify SIN's metabolites. Enzymatic and non-enzymatic incubations were used to reveal SIN's metabolic mechanism. Impacts of SIN, SNO and DS on the inflammation-related cytokine's levels and nuclear translocation of NF-κB were evaluated in LPS-induced Raw264.7 cells. ROS induced by SNO (10 μM) was also assessed. Key findings: CYP3A4 and ROS predominantly mediated the formation of SNO, and CYP3A4 and CYP2C19 primarily mediated the formation of DS. Noteworthily, SNO underwent N-oxide reduction both enzymatically, by xanthine oxidase (XOD), and non-enzymatically, by ferrous ion and heme moiety. The levels of IL-6 and TNF-α and nuclear translocation of NF-κB were ameliorated after pretreatment of SIN in LPS-induced Raw264.7 cells, while limited attenuations were observed after pretreatment of DS (SNO) even at 200 μM. In contrast, SNO induced ROS production. Significance: This study elucidated that SIN underwent both enzymatic and non-enzymatic cyclic metabolism and worked as the predominant anti-inflammation compound, while SNO induced ROS production, suggesting more studies of SIN combined with SNO and DS are necessary in case of DDI and potential toxicities.

Full text of DOI:10.1016/j.lfs.2020.118433

LifeSciences261(2020)118433
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Metabolic mechanism and anti-inflammation effects of sinomenine and its
major metabolites N-demethylsinomenine and sinomenine-N-oxide
⁎
⁎⁎
,1
Qiang Lia, Wenbin Zhoua,b, Yuyan Wanga, Fang Koua, Chunming Lyua,b, ,1, Hai Weia,
a Institute of Interdisciplinary Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
b Shanghai Zhulian Intelligent Technology Co., Ltd., Shanghai, China
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article:
Sinomenine
Aims: Sinomenine (SIN) is clinically used as an anti-rheumatic drug. However, the metabolic and pharmaco-
logical mechanisms of SIN combined with its metabolites are unclear. This study aims to explore the cyclic
metabolic mechanism of SIN, the anti-inflammation effects of SIN and its major metabolites (N-demethylsino-
menine (DS) and sinomenine-N-oxide (SNO)), and the oxidation property of SNO.
N-demethylsinomenine
Sinomenine-N-oxide
Lipopolysaccharide
Materials and methods: SIN was administrated to rats via gavage. Qishe pills (a SIN-containing drug) were orally
administrated to humans. The bio-samples were collected to identify SIN's metabolites. Enzymatic and non-
enzymatic incubations were used to reveal SIN's metabolic mechanism. Impacts of SIN, SNO and DS on the
inflammation-related cytokine's levels and nuclear translocation of NF-κB were evaluated in LPS-induced
Raw264.7 cells. ROS induced by SNO (10 μM) was also assessed.
Dexamethasone
Keywords:
Sinomenine
N-demethylsinomenine
Sinomenine-N-oxide
CYP450s
Key findings: CYP3A4 and ROS predominantly mediated the formation of SNO, and CYP3A4 and CYP2C19
primarily mediated the formation of DS. Noteworthily, SNO underwent N-oxide reduction both enzymatically,
by xanthine oxidase (XOD), and non-enzymatically, by ferrous ion and heme moiety. The levels of IL-6 and TNF-
α and nuclear translocation of NF-κB were ameliorated after pretreatment of SIN in LPS-induced Raw264.7 cells,
while limited attenuations were observed after pretreatment of DS (SNO) even at 200 μM. In contrast, SNO
induced ROS production.
Heme moiety
Anti-inflammation effects
ROS (reactive oxygen species)
Significance: This study elucidated that SIN underwent both enzymatic and non-enzymatic cyclic metabolism
and worked as the predominant anti-inflammation compound, while SNO induced ROS production, suggesting
more studies of SIN combined with SNO and DS are necessary in case of DDI and potential toxicities.
1. Introduction
RA [5] due to its evident anti-inflammation effects [6,7]. During the
inflammation, overproduction of nitric oxide (NO), tumor necrosis
Sinomenine (SIN) is an alkaloid derived from the Chinese herb
Sinomenium acutum (Thunb.) Rehd. et Wils [1]. SIN is clinically used in
China with a trade name Zhengqing Fengtongning (FTN). FTN injec-
tion, FTN tablet and FTN sustained-release tablet have been approved
by the State Food and Drug Administration of China to treat rheumatoid
arthritis (RA). RA is a chronic disease characterized as a long-term,
progressive, and disabling autoimmune disease [2]. Systemic in-
flammation in joints is the significant symptom of RA. Statistically, RA
affects up to 1% of the population worldwide, with females being much
more susceptible [3]. Based on the clinical trials, the efficacy rate of SIN
to treat RA is about 90% [4]. SIN is recommended as a substitutive drug
to non-steroidal anti-inflammatory drugs (NSAIDs) for the treatment of
factor (TNF-α) and interleukin-6 (IL-6) by T cells and macrophages are
always involved [8–10]. SIN can significantly decrease the level of
iNOS, TNF-α and IL-6 against the inflammation. For the mechanism,
down-regulation of CCAT1 (long non-coding RNA colon cancer asso-
ciated transcript-1) was verified to contribute to its anti-inflammation
activity [6]. The inhibition of the ERK/Egr-1 signal pathway was also
involved in the anti-inflammation mechanism of SIN [11].
The anti-inflammation effects of SIN have been widely evaluated
and confirmed, whereas some essential issues are still unclear. Is SIN
stable enough with sufficient systemic exposure to manifest the anti-
inflammation effects in vivo after its administration? If not, do either or
both of SIN and its major metabolites mediate the pharmacological
⁎ Correspondence to: C. Lyu, Shanghai Zhulian Intelligent Technology Co., Ltd., Shanghai, China.
⁎⁎ Corresponding author.
E-mail addresses: chunming83g@126.com (C. Lyu), wei_hai@hotmail.com (H. Wei).
1 There corresponding authors contributed equally to this work.
https://doi.org/10.1016/j.lfs.2020.118433
Received 31 July 2020; Received in revised form 30 August 2020; Accepted 9 September 2020
Availableonline17September2020
0024-3205/©2020ElsevierInc.Allrightsreserved.

Q. Li, et al.
LifeSciences261(2020)118433
Fig. 1. Chemical structure of sinomenine (SIN), N-demethyl-sinomenine (DS) and sinomenine-N-oxide (SNO).
effects in vivo? Besides, the side effects of SIN [5,12] and the drug-drug
interactions (DDI) related to SIN should not be ignored [13,14]. As is
reported, during multi-dose administration of SIN tablet, low oral
bioavailability and high fluctuation of plasma concentration were ob-
served, which may relate to the occasional gastrointestinal tract toxicity
and allergic reactions [13,15]. Thus, the pharmacokinetics of SIN,
especially its metabolism, are fundamental, whereas the relevant stu-
dies are quite limited. Although two metabolites with [M + H]+ ion
peak at 316 and 346 were observed after the administration of SIN to
humans, the chemical structures were not identified [16]. N-de-
methylsinomenine (DS, [M + H]+ at 316) was firstly isolated from
incubations of human liver microsomes with SIN in vitro by Christians
Uwe (Fig. 1) [17]. Sinomenine-N-oxide (SNO, [M + H]+ at 346) was
firstly identified in rat urine by Cheng WM (Fig. 1) [18]. Yet, no sys-
temic research is performed to reveal the cyclic metabolic mechanism
of SIN in humans. Besides, the anti-inflammation effects and possible
toxicities of SIN's major metabolites are still unclear.
of the compounds using dichloromethane (30 mL) three times. The
water in the extraction solution was removed using anhydrous sodium
sulfate. SNO (purity > 99%) was then collected by evaporation of the
filtrate.
Synthesis of DS: about 100 mg of SNO was dissolved in 15 mL of
methanol, followed by the addition of FeSO4·7H2O (100 mg) with
stirring overnight at room temperature. Thereafter, the supernatant was
evaporated to dryness, followed by the addition of EDTA solution
(0.06 mmol/mL, 20 mL). The pH value was adjusted to 10 after four-
hour stirring, and the mixture was extracted using dichloromethane
(30 mL) three times. Anhydrous sodium sulfate was added into the
extraction solution to remove the water, followed by the evaporation of
the filtrate to obtain the crude product of DS. The purified DS was se-
parated by semi-preparation HPLC with a final purity higher than 99%.
2.2. Characterization of the major metabolites of SIN in vivo
To explore the major metabolites of SIN with elucidation of the
cyclic metabolic mechanism, metabolic analysis of in-vivo bio-samples
and in-vitro enzymatic and non-enzymatic incubations were performed.
Meanwhile, the anti-inflammation effects of SIN, SNO and DS and the
oxidation property of SNO in LPS-induced Raw264.7 cells were eval-
uated in the current study.
2.2.1. Characterization of the major metabolites of SIN in rats
Adult male Sprague-Dawley rats (200–250 g) were obtained from
Sino-British Sippr/BK Laboratory Animal Ltd. (China). The animals
were housed with free access to water. The jugular vein of the rat was
cannulated for the collection of plasma. Additionally, the bile duct of
the rat was cannulated. All animal procedures were performed in ac-
cordance with the Animal Ethics Committee guidelines under the ap-
proval of Animal Ethics Committee of Shanghai University of
Traditional Chinese Medicine.
2. Materials and methods
Plasma, urine, and bile sample collections: SIN was dissolved in
water and then administrated to rat via gavage at a dose of 50 mg/kg.
Blood samples (200 μL at 0, 1, 2, 4, 8, 24 and 48 h) were collected via
the jugular vein catheter. The plasma was obtained by centrifugation of
the blood at 4000 ×g for 5 min. Additionally, urine samples were
collected at different time intervals of 0 h, 0–4 h, 4–8 h, 8–12 h,
12–24 h and 24–48 h. Bile samples were collected at 0 h and 0–6 h.
2.1. Materials
For metabolism study: Male and female human liver microsomes
(MHLMs/FHLMs), rat liver microsomes (MRLMs/FRLMs), and NADPH
were purchased from XenoTech, LLC (USA). NADH was obtained from
Roche Diagnostics GmbH (Germany). Purified recombinant human
CYP450 isozymes (CYP1A1, CYP1A2, CYP2A6, CYP3A4, CYP3A5,
CYP2B6, CYP2C9, CYP2C19, CYP2D6*2, CYP2D6*10 and CYP2E1)
were obtained from Cypex Ltd. (Scotland). Propranolol (Internal stan-
dard, I.S.), catalase (CAT), hemoglobin, and xanthine were obtained
from Sigma-Aldrich (USA). 7-hydroxycoumarin was supplied from
Sinopharm Chemical Reagent Co. Ltd. (China). SIN was purchased from
National Institutes for Food and Drug Control (China). Superoxide
Dismutase (SOD) kits were purchased from Nanjing Jiancheng
Bioengineering Institute (China). Methanol was supplied by Thermo
Fisher Scientific (USA).
2.2.2. Testification of the major metabolites of SIN in humans
SIN is one of the major ingredients in the Qishe pill (26 mg of SIN in
a single dose of the Qishe pill [19]). A single-center, open-label, double
Latin-sequence, and crossover study was conducted in healthy adult
subjects [20]. This clinical trial was approved by the ethics committee
in Longhua Hospital affiliated to Shanghai University of Traditional
Chinese Medicine (2013-LCSY-064) and took place at Longhua Hospital
at the phase I unit (ClinicalTrials.gov Identifier: NCT02294448). The
work has been carried out in accordance with the Declaration of Hel-
sinki for all the experiments involving humans. The bio-samples after
oral administration of the Qishe pill (single dose) were collected based
on the documents.
For
anti-inflammation
and
oxidation
property
study:
Lipopolysaccharide (LPS) and dexamethasone (DEX) were purchased
from Sigma Aldrich (USA). HRP-labeled goat anti-rabbit IgG was pur-
chased from Beyotime Biotechnology (China). IL-6 and TNF-α assay kits
were purchased from Nanjing Jiancheng Bioengineering Institute
(China). ROS (Reactive Oxygen Species) assay kits were obtained from
Beyotime Biotechnology (China).
2.2.3. Sample preparation
Preparation of the plasma and urine sample: An aliquot of 800 μL of
methanol was added into 200 μL of plasma or urine, followed by vortex
mixing for 3 min. After centrifugation of the mixture at 16,000 × g for
15 min, an aliquot of 700 μL of the supernatant was evaporated to
Synthesis of SNO: about 0.8 g of SIN was added into 15 mL of 30%
H2O2, followed by stirring the mixture at room temperature for 60 h.
Thereafter, the pH value was adjusted to 10, followed by the extraction
2

Q. Li, et al.
LifeSciences261(2020)118433
dryness and reconstituted by 140 μL of 30% methanol. The supernatant
after centrifugation was injected into the UPLC-MS/MS for the analysis
of SIN and its potential metabolites.
different groups.
2.3.5. CYP450 enzyme kinetics of SIN for the formation of DS and SNO
Incubation mixture (100 μL) contained enzymes, serial concentra-
tions of SIN, and MgCl2 (3.3 mM) in potassium phosphate buffer
(100 mM, pH 7.4). The optimized protein concentrations of HLMs,
RLMs, CYP2C19 and CYP3A4 were at 0.5 mg/mL. The reactions were
initiated by the addition of NADPH (1 mM) and incubated at 37 °C for
60 min for HLMs (20 min for RLMs, CYP2C19 and CYP3A4). Thereafter,
aliquots of 600 μL of ice-cold I.S.-containing methanol were added into
the incubations. The formation rate of DS or SNO was plotted versus the
SIN substrate concentrations. The kinetic parameters were calculated
by GraphPad Prism software. The best kinetic model was selected based
on the Eadie-Hofstee plot [24].
Preparation of the bile sample: An aliquot of 4 μL of the bile sample
was diluted to 10 mL by methanol. After 15-min centrifugation at
16,000 × g, the supernatant was injected into UPLC-MS/MS for the
analysis of SIN and its potential metabolites.
2.3. Verification of SIN's major metabolites in vitro and exploration of SIN's
metabolic mechanism
2.3.1. Stability of SIN in different kinds of buffers, GI content and biofluids
Stability of SIN in buffers (pH at 1.2, 6.0 and 7.2), SGF (simulated
gastric fluid), SIF (simulated intestinal fluid), GI content, plasma, urine,
and bile were performed. The gastric content and intestinal content of
rats were prepared based on the previous study [21]. An aliquot of
10 μL of SIN (33 mM) was incubated with 990 μL of the above medium
for 120 min, respectively. Aliquots of 100 μL of samples were collected
at different time intervals of 0, 10, 60 and 120 min, followed by the
addition of 600 μL of ice-cold methanol.
2.3.6. Sample preparation
The terminated incubation samples were centrifugated at
16,000 ×g for 15 min. Thereafter, the supernatant (300 μL) was col-
lected and evaporated under nitrogen. The residue was reconstituted by
100 μL of 30% methanol. After 15-min centrifugation of the mixture at
16,000 × g, the resulting supernatant was analyzed by UPLC-MS/MS.
2.3.2. Verification of SIN's metabolites using male and female HLMs
(RLMs)
2.4. Further exploration of the metabolism of DS and SNO
To further verify SIN's major metabolites identified in humans and
rats in vivo, incubations of HLMs (RLMs) with SIN were performed. SIN
was incubated with HLMs (RLMs) in triplicate at 37 °C for 120 min. The
mixture (100 μL) contained HLMs (1 mg/mL) or RLMs (1 mg/mL),
MgCl2 (3.3 mM), and SIN (3.3 μM) in potassium phosphate buffer
(100 mM, pH 7.4) in the absence or presence of NADPH (1 mM). The
reactions were terminated by the addition of 600 μL of ice-cold me-
thanol.
2.4.1. Stability of DS and SNO
In vitro stability of DS and SNO: Stabilities of DS and SNO in plasma,
urine, and bile were performed. DS or SNO (10 μM, 10 μL) was in-
cubated with 990 μL of plasma, urine, and bile for 120 min, respec-
tively. Aliquots of 100 μL of samples were collected at 0 min, 10 min,
60 min and 120 min, followed by the addition of 600 μL of methanol.
In vivo metabolism of DS and SNO: DS (6.67 mg/kg) and SNO
(6.67 mg/kg) were administrated to male Sprague-Dawley rats via ga-
vage, respectively. The bile and urine samples were collected at dif-
ferent time intervals of 0 h, 0–2 h and 2–6 h. Aliquots of 600 μL of
methanol were added into the samples (100 μL).
2.3.3. Characterization of the CYP450 isozymes mediating the formation of
DS and SNO
The recombinant human CYP450 isozymes were used to identify the
specific CYP450 isozymes mediating the formation of DS and SNO. The
incubation mixture (100 μL) consisted of recombinant human CYP450
isozymes (1 mg/mL), MgCl2 (3.3 mM), SIN (3.3 μM), and NADPH
(1 mM) in potassium phosphate buffer (100 mM, pH 7.4). The reactions
were terminated after 60-min incubation (37 °C) by the addition of
600 μL of ice-cold I.S.-containing methanol. The formation rate of DS or
SNO, expressed as the amount of DS or SNO generated per min per
milligram protein, was calculated.
Sample preparation: After centrifugation of the mixture, the super-
natant (600 μL) was collected and evaporated under nitrogen. The re-
sidue was reconstituted by 100 μL of 30% methanol. After 15-min
centrifugation of the mixture at 16,000 × g, the resulting supernatant
was analyzed using UPLC-MS/MS.
2.4.2. Further identification of enzymatic and non-enzymatic metabolic
pathways of SNO
N-oxide reduction of SNO in HLMs and HLCs (non-enzyme): The
incubation mixture (100 μL) was composed of HLMs (1 mg/mL) or
HLCs (1 mg/mL), SNO (10 μM), and electron donor in potassium
phosphate buffer (100 mM, pH 7.4). The electron donor was NADPH
(1 mM), NADH (2 mM) or FAD (0.1 mM). Sodium azide (100 nM) was
used as an inhibitor of electron transfer. The anaerobic incubations
were performed by pre-gassing of nitrogen for 2 min. The reaction was
initiated by the addition of SNO (10 μM) with a subsequent 120-min
incubation at 37 °C. The assay was also performed using denatured
HLMs (HLCs) instead of the native HLMs (HLCs) by heating them in a
boiling water bath for 5 min.
2.3.4. Characterization of the role of ROS in the formation of SNO
XOD catalyzes the metabolism of hypoxanthine to xanthine and
xanthine to uric acid with the formation of ROS [22]. The incubation
mixture (100 μL) consisted of xanthine (0.3 mM), XOD (6.5 munits/mL)
and SIN (33.3 μM). The reactions initiated by the addition of xanthine
at 37 °C. The control test was performed in the absence of XOD. The
negative test was performed in the absence of xanthine. In detail, three
kinds of studies were performed. A: The incubations of XOD (6.5 mu-
nits/mL) with SIN (33.3 μM) in the presence of xanthine (0.3 mM) were
performed at 37 °C for 0, 5, 10, 15 and 20 min, respectively. B: The
incubations of XOD (1-fold (6.5 munits/mL) and 4-fold (26 munits/
mL)) with serial concentrations of SIN (8.3, 16.7, 33.3, 66.7 and
133.3 μM) in the presence of xanthine (0.3 mM) were performed at
37 °C for 20 min, respectively. C: For the Reaction group, incubations of
XOD (6.5 munits/mL) with SIN (33.3 μM) in the presence of xanthine
(0.3 mM) were performed at 37 °C for 20 min. For the Reaction + CAT
group, incubations were performed with the addition of catalase
(475 units/mL) [23] in comparison to the Reaction group. For the Re-
action + SOD group, incubations were performed with the addition of
SOD (75 units/mL) [23] in comparison to the Reaction group. The
samples were prepared by the addition of 600 μL of ice-cold I.S.-con-
taining methanol. The formation rate of SNO was compared among
N-oxide reduction of SNO in HLCs (enzyme): The incubation mix-
ture (100 μL) was composed of HLCs (1 mg/mL), SNO (10 μM), and
xanthine (1 mM) in potassium phosphate buffer (100 mM, pH 7.4). In
the inhibition study, 7-hydroxycoumarin was used as an inhibitor of
XOD [25]. The anaerobic incubations were performed by pre-gassing of
nitrogen for 2 min. The reaction was initiated by the addition of SNO
(10 μM) with a subsequent 120-min incubation at 37 °C. The parallel
incubations were also performed using denatured HLCs.
N-oxide reduction of SNO in denatured catalase, denatured he-
moglobin, and ferrous ion: Incubation mixture (100 μL) contained de-
natured catalase (1 mg/mL) or denatured hemoglobin (1 mg/mL), SNO
(10 μM), and electron donor in potassium phosphate buffer (100 mM,
3

Q. Li, et al.
LifeSciences261(2020)118433
pH 7.4). The electron donor was NADPH (1 mM), NADH (2 mM) or FAD
(0.1 mM). The anaerobic incubations were performed by pre-gassing of
nitrogen for 2 min. The reaction was initiated by the addition of SNO
with a subsequent 120-min incubation at 37 °C. The role of ferrous ion
in the metabolism of SNO, with an identical concentration of the ferrous
ion in hemoglobin, was also evaluated.
diacetate (DCFH-DA) assay [26]. Raw264.7 cells were seeded in 6-well
plates (1 × 106 cells/well). Cells were incubated for 12 h. Subse-
quently, H2O2 (100 μM) and SNO (10 μM) were incubated with
Raw264.7 cells for 120 min, respectively. Thereafter, the cells were
rinsed three times, followed by the incubation with LPS (0.1 μg/mL) for
24 h. DCFH-DA (10 μM) was then added to each well after the rinse of
the cells, followed by the incubation for 0.5 h. Then, the cells were
collected to determine the fluorescence intensity using a flow cytometer
(Attune NXT, Thermo, USA).
Sample preparation: All the samples were prepared immediately by
the addition of 600 μL of ice-cold I.S.-containing methanol after in-
cubations for 120 min, followed by 3-min vortex mixing and cen-
trifugation at 16,000 ×g for 15 min. The supernatant (300 μL) was
collected and evaporated under nitrogen. The residue was reconstituted
by 100 μL of 30% methanol. After 15-min centrifugation of the mixture
at 16,000 × g, the resulting supernatant was analyzed by UPLC-MS/MS
to quantify the content of SIN formation.
2.6. LC-MS/MS analyses
2.6.1. Scan mode for preliminary identification of the metabolites of SIN
An Agilent 6470 triple quadruple mass spectrometer (Agilent
Technologies, USA) coupled with electrospray ionization (ESI) source
was employed for preliminary identification of the metabolites of SIN.
Chromatographic separations were achieved by a Waters ACQUITY
UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm particles). Mobile
phases consisted of eluents A (0.1% formic acid in water) and B (me-
thanol). A 1-μL aliquot of the sample was injected into and eluted off
the column at a flow rate of 0.3 mL/min. The gradient elution program
was described as follows: 0–1.0 min (3% B), 1.0–10.0 min (3–30% B),
10.0–12.0 min (30–60% B), 12.0–13.0 min (60–3% B), and 13.0–15.0
(3% B). The temperatures of the auto-sampler and column were set at
8 °C and 30 °C, respectively. The optimized MS parameters were shown
as follows: positive-ion mode, gas temperature at 300 °C, gas flow at
5 L/min, nebulizer gas with 45 psi, sheath gas temperature at 250 °C,
sheath gas flow at 11 L/min, nozzle voltage at 500 V, source voltage at
3500 V, and nozzle voltage at 500 V.
2.5. Exploration of the anti-inflammation effects of SIN, DS and SNO and
oxidation property of SNO
2.5.1. Cell culture
Raw 264.7 cells were incubated in DMEM supplemented with 10%
FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin in a humidified
incubator with 5% CO2 and 95% air at 37 °C.
2.5.2. Measurement of IL-6 and TNF-α protein levels by ELISA
RAW 264.7 cells were seeded in 6-well plates (about 1 × 106 cells/
well) and cultured for 12 h, followed by pretreatment with SIN (10, 100
and 200 μM), SNO (10, 100 and 200 μM), DS (10, 100 and 200 μM) and
DEX (25 μM) for 120 min, respectively. Thereafter, LPS (0.1 μg/mL)
was added to the medium with a subsequent incubation for 24 h. The
medium was collected and centrifuged, followed by the determination
of the cytokines using IL-6 and TNF-α ELISA kits based on the manu-
facturer's protocols.
2.6.2. Multiple reaction monitoring (MRM) Mode for quantification of SIN,
DS and SNO
2.5.3. Analysis of TNF-α mRNA levels by RT-PCR
RAW 264.7 cells were seeded in 6-well plates (about 1 × 106 cells/
well) and cultured for 12 h, followed by pretreatment with SNO
(200 μM), DS (200 μM) and DEX (25 μM) for 120 min, respectively. LPS
at 0.1 μg/mL was added to the medium, followed by incubation for
24 h. Total RNA was extracted with TRIzol Reagent, and M-MLV reverse
transcriptase was used to reversely transcribe RNA using random hex-
amers at 37 °C. The diluted transcribed cDNA was mixed with gene-
specific primer and SYBR Green. Changes in gene expression were
analyzed using an RT-PCR machine (QuantStudio 6 Flex System,
Applied Biosystems, USA). The 2−ΔΔCT method was used to calculate
the fold-change of gene expression with GAPDH as the housekeeping
gene. The sequences of primers used were as follows: GAPDH: AGGT
CGGTGTGAACGGATTTG (F) and GGGGTCGTTGATGGCAACA (R);
TNF-α: CAGGCGGTGCCTATGTCTC (F) and CGATCACCCCGAAGTTCA
GTAG (R).
A series 1290 UPLC system coupled with an Agilent 6470 triple
quadruple mass spectrometer (ESI source) was employed for quantifi-
cation of SIN, SNO and DS. Chromatographic separations were achieved
by a Waters ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm
particles). Mobile phases consisted of eluents A (0.1% formic acid in
water) and B (methanol). A 2-μL aliquot of the sample was injected into
and eluted off the column at a flow rate of 0.3 mL/min. The gradient
elution program was described as follows: 0–1.0 min (10% B),
1.0–1.1 min (10–20% B), 1.1–2.0 min (20% B), 2.0–2.1 min (20–30%
B), 2.1–3.0 (30% B), 3.0–3.5 min (30–80% B), 3.5–4.0 (80% B),
4.0–4.5 min (80–10% B), and 4.5–6.0 min (10% B). The temperatures of
the auto-sampler and column were set to be 8 °C and 30 °C, respec-
tively. The optimized MS parameters were shown as follows: positive-
ion mode, gas temperature at 300 °C, gas flow at 5 L/min, nebulizer gas
with 45 psi, sheath gas temperature at 250 °C, sheath gas flow at 11 L/
min, nozzle voltage at 500 V, source voltage at 3500 V, and nozzle
voltage at 500 V. The MRM mode was adopted for quantification of all
the analytes. In detail, SIN: m/z 330.4 → 207.2, Fragmentor 190 V,
Collision energy 38; SNO: m/z 346.2 → 314.2, Fragmentor 135 V,
Collision energy 30; DS: m/z 316.1 → 239.2, Fragmentor 150 V,
Collision energy 30; Propranolol (I.S.): m/z 260.2 → 116.3, Fragmentor
110 V, Collision energy 14.
2.5.4. Evaluation of the nuclear translocation of NF-kB p65 by western
blotting
The cells mentioned in Section 2.5.2 were washed twice. The total
protein was extracted with RIPA lysate buffer with the determination of
the protein concentrations. Equal amounts (10 μg/lane) of the protein
samples were separated by SDS-PAGE and then transferred onto PVDF
membranes. Subsequently, the membranes were incubated with pri-
mary antibodies and anti-GAPDH overnight at 4 °C after the incubation
of the membranes with 5% BSA for 120 min. After incubation with
horseradish peroxidase-conjugated goat anti-rabbit IgG secondary an-
tibody for 4 h at room temperature, the blots were visualized using an
enhanced chemiluminescent ECL reagent. The protein bands were
quantified by the ChemiDoc XRS gel imaging system (Bio-Rad, USA).
2.7. Statistical analysis
Data were expressed as mean
SD. Statistical analysis was per-
formed using GraphPad Prism software (USA). One-way analysis of
variance (ANOVA) followed by Dunnett's multiple comparisons test was
used to assess the significance among groups. p < 0.05 was considered
statistically significant.
2.5.5. Exploration of the oxidation property of SNO
Intracellular ROS was measured using the 2,7-dichlorofluorescin
4

Q. Li, et al.
LifeSciences261(2020)118433
Fig. 2. UPLC-MS/MS extracted total ion chromatograms of potential metabolites of SIN in different samples. A. Blank urine sample of rats. B. Urine sample of rats at
8–12 h after the administration of SIN via gavage (50 mg/kg). C. Blank urine sample of humans. D. Urine sample of humans at 8–12 h after oral administration of a
single dose of the Qishe pill (containing 26 mg of SIN). Human Urine: Human urine sample (8–12 h) after oral administration of the Qishe pill. Rat Urine: Rat urine
sample (8–12 h) after the administration of SIN via gavage. Human Plasma: Human plasma sample (2 h) after oral administration of the Qishe pill. Rat Plasma: Rat
plasma sample (2 h) after the administration of SIN via gavage. Rat Bile: Rat bile sample (0–6 h) after the administration of SIN via gavage. Heatmap shows the ratio
(log10 transformation) of each metabolite's response versus SIN's response in different samples. Shades of red and blue represent the high ratio and the low ratio of a
metabolite's formation in comparison to SIN, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
3. Results
the presence of NADPH were performed to verify the formation of DS
and SNO. As it turned out, considerable formations of DS and SNO were
observed.
3.1. Identification and verification of the major metabolites of SIN
3.1.1. Screening the major metabolites of SIN in vivo
3.2. Enzymatic and non-enzymatic metabolism of SIN via CYP450s and
ROS
In comparison to the blank urine sample of rats (Fig. 2A), ten me-
tabolites were found in rats after the administration of SIN via gavage
(Fig. 2B). The metabolite with protonate molecular weight at m/z 316.4
(M1, parent −14) was eluted at 6.8 min, inferring that it may be a
demethyl metabolite of SIN. The metabolites, namely M2-1, M2-2 and
M2-3, with identical protonated ions at m/z 332.3 (parent +2), were
eluted at 5.5, 6.5 and 6.9 min, indicating a probable addition of two
hydrogens into the molecule of SIN. Another kind of metabolites (M3-1,
M3-2, M3-3, M3-4 and M3-5) had the same protonate molecular weight
at m/z 346.4 (parent +16) with retention time at 3.3, 4.0, 5.6, 6.7 and
6.9 min, respectively, suggesting a potential addition of one oxygen
atom into the molecule of SIN. Besides, another metabolite with re-
tention time at 9.9 min was observed (M4 with m/z at 362.4, parent
+32).
3.2.1. Characterization of CYP450 isozymes mediating the formation of
SNO and DS
As shown in Fig. 3, CYP3A4 and CYP2C19 were identified as the
predominant CYP450 isozymes mediating the formation of DS, and
CYP3A4 was the major CYP450 isozyme mediating the formation of
SNO.
3.2.2. Evidence of ROS mediating the formation of SNO
A trace amount of SNO was observed even if SIN was incubated with
denatured liver microsomes, indicating non-enzymatic metabolism may
play a role in the formation of SNO. Due to the existence of ROS in live
microsomes [27]. We hypothesized that SIN may react with ROS to
generate SNO. SNO cannot be observed in the negative, control and 0-
min incubations (Fig. 4A and D). There was a positive correlation be-
tween the increased formation of SNO and the increased production of
ROS (formation of ROS increased with time during the 20-min in-
cubations), indicating ROS may partly mediate the metabolism of SIN
with the formation of SNO. Additionally, to synthesize SNO in vitro, SIN
was incubated with 30% H2O2 at room temperature for 60 h with a
transformation rate (SIN to SNO) at about 100%, suggesting that SIN
can react with H2O2 to generate SNO. As shown in Fig. 4B, a 4-fold
concentration of XOD was used in the incubations to increase the for-
mation of ROS. Results manifested that the formation of SNO sig-
nificantly increased with the increment of ROS, indicating ROS reacted
with SIN to generate SNO. Moreover, catalase was used to eliminate
H2O2, and SOD was used to eradicate O2- (Fig. 4C). As shown in Fig. 4D,
only O2- existed in the Reaction + CAT group, and only H2O2 existed in
the Reaction + SOD group, whereas SNO was still observed, inferring
both O2- and H2O2 participated in the metabolism of SIN with the
formation of SNO.
As shown in Fig. 2, the heatmap was plotted to manifest the ratio
(log10 transformation) of each metabolite's response versus SIN's re-
sponse in different samples. Since the other components in the Qishe
pill may hamper the analysis of the metabolites derived from SIN, the
metabolites were firstly identified from the bio-samples of rats and then
testified from the bio-samples of humans. Most of the SIN's metabolites
found in rats were positively observed in the urine and plasma of hu-
mans. Based on the heatmap, M1 and M3-4 were found to be the most
predominant metabolites in humans and rats in vivo. Thus, M1 and M3-
4 were synthesized for further study. The chemical structures of DS and
SNO were confirmed by NMR data (Supplemental 1) [18]. M1 was
identified as N-demethylsinomenine (DS), and M3-4 was identified as
sinomenine-N-oxide (SNO). The ratio of SNO:DS:SIN in human urine
(8–12 h) after oral administration of Qishe pills is 0.3: 0.02: 1, and the
ratio of SNO:DS:SIN in rat urine (8–12 h) after oral administration of
SIN is 0.01: 0.7: 1. Besides, five metabolites of SIN with [M + H]+ at
346.4 (SNO and another four potential hydroxylated metabolites of
SIN) were observed in rats. Yet, only one metabolite of SIN with
[M + H]+ at 346.4 (SNO) was found in humans, inferring the existence
of species differences in the metabolism of SIN.
3.2.3. CYP450 enzyme kinetics for the formation of DS and SNO
For the formation of DS, the substrate inhibition equation best fitted
the data obtained from the incubations using FHLMs, MHLMs, FRLMs,
MRLMs, CYP2C19 and CYP3A4 based on the Eadie-Hofstee plots
(shown in Fig. 5). It was observed that Clint in MHLMs was significantly
higher than that in FHLMs (Table 1). In the incubations of FRLMs and
3.1.2. Verification of the major metabolites of SIN in vitro
SIN was stable in 120-min incubations in different kinds of medium,
including buffers (pH at 1.2, 6.0 and 7.2), SGF, SIF, GI content, plasma,
urine, and bile. Furthermore, incubations of HLMs (RLMs) with SIN in
5

Q. Li, et al.
LifeSciences261(2020)118433
Fig. 3. Characterization of CYP450 iso-
zymes mediating the formation of DS and
SNO. The incubation mixture (100 μL)
consisted of recombinant human CYP450
isozymes (CYP1A1, CYP1A2, CYP2A6,
CYP3A4, CYP3A5, CYP2B6, CYP2C9,
CYP2C19, CYP2D6*2, CYP2D6*10 or
CYP2E1 at 1 mg/mL), MgCl2 (3.3 mM), SIN
(3.3 μM), and NADPH (1 mM) in potassium
phosphate buffer (100 mM, pH 7.4). The
incubations were performed at 37 °C for
60 min. The formation rate of DS or SNO,
expressed as the amount of DS or SNO
generated per min per milligram protein,
was calculated. Each data point is expressed
as mean
SD of triplicates.
Fig. 4. Characterization of ROS mediating the formation of SNO. A. The concentrations of SNO generated in incubations of XOD (6.5 munits/mL) with SIN (33.3 μM)
in the presence of xanthine (0.3 mM) at 37 °C for 0, 5, 10, 15, and 20 min. B. The concentrations of SNO generated in incubations of XOD (1-fold (6.5 munits/mL) and
4-fold (26 munits/mL)) with serial concentrations of SIN (8.3, 16.7, 33.3, 66.7 and 133.3 μM) in the presence of xanthine (0.3 mM) at 37 °C for 20 min. C. The
schematic diagram of ROS production and ROS transformation (XOD: xanthine oxidase; CAT: catalase; SOD: superoxide dismutase). D. The concentrations of SNO
generated in incubations of XOD (6.5 munits/mL) with xanthine (0.3 mM) and SIN (33.3 μM) in the absence or presence of CAT or SOD for 20 min. Each data point is
expressed as mean
SD of triplicates. **p < 0.01, ***p < 0.001, significant difference vs. the reaction group.
6

Q. Li, et al.
LifeSciences261(2020)118433
7

Q. Li, et al.
LifeSciences261(2020)118433
Table 1
Parameters of enzyme kinetics of SIN for the formation of DS and SNO.
Vmax (pmol/min/mg)
Km (μM)
Clint (μL/min/mg)
Ki (μM)
R2
Type
DS formation
MHLMs
227
231
(2.91
268
617
516
7
1.41
1.74
1.44
1.76
1.24
2.41
0.08
0.31
0.14
0.61
0.58
0.30
161
137
(2.03
152
500
214
4
31.9
16.0
14.0
17.3
21.5
20.6
3.4
3.8
1.8
7.8
1.5
3.9
0.996
0.963
0.985
0.925
0.997
0.989
Substrate inhibition
Substrate inhibition
Substrate inhibition
Substrate inhibition
Substrate inhibition
Substrate inhibition
FHLMs
22
12⁎
MRLMs
0.15) × 103
0.97) × 103
FRLMs
49
14
37
21###
CYP3A4
7
3
CYP2C19
SNO formation
MHLMs
202
294
501
4
1.09
1.24
3.08
0.08
0.23
0.57
194
205
172
10
39
12
–
0.982
0.949
0.959
Michaelis-Menten
Substrate inhibition
Michaelis-Menten
FHLMs
26
31
32.8
–
11.1
CYP3A4
Each data point is expressed as mean
SD of triplicates.
⁎
p < 0.05, significant difference vs. MHLMs.
###
p < 0.001, significant difference vs. MRLMs.
MRLMs with SIN, the Clint of SIN for the formation of DS in MRLMs was
about 13-fold higher in comparison to that in FRLMs. For the formation
of SNO, the substrate inhibition equation best fitted the data obtained
from the incubations using MHLMs, whereas typical Michaelis-Menten
equation best fitted the data collected from incubations using FHLMs
and CYP3A4. The relevant parameters were shown in Table 1. The Clint
of SIN for the formation of SNO was slightly higher than that of DS in
HLMs.
Table 2
Enzymatic and non-enzymatic N-oxide reduction of SNO in HLMs and HLCs.
Addition
SIN formation (pmol/min/mg)
Native enzyme
Boiled enzyme
HLMs (non-enzyme)
None (negative incubation)a
None (negative incubation)b
NADH
N.D.
–
12.9
17.5
20.7
10.8
0.9
1.9
1.8
1.7
12.2
19.6
16.8
10.6
1.3
4.0
2.9
1.5
NADPH
3.3. Further metabolism of DS and SNO with a unique metabolic
mechanism
NADH, NaN3
HLCs (non-enzyme)
None (negative incubation)c
None (negative incubation)d
NADH
N.D.
23.3
23.9
28.9
38.3
28.0
–
3.3.1. Stability of DS and SNO
0.2
0.3
0.5
2.0
1.2
22.0
22.8
28.0
38.6
31.6
2.0
1.5
1.5
2.3
2.1
DS and SNO were stable in the 120-min in-vitro incubations with
plasma, urine, and bile. After oral administration of DS to rats, no ob-
vious metabolite was observed in urine and bile. However, plenty of
SIN with a trace amount of DS was found in bile after the administration
of SNO to rats, indicating SNO can be metabolized into SIN and DS in
vivo.
NADPH
NADH and FAD
NADH, FAD and NaN3
HLCs (enzyme)
Xanthine
28.4
21.9
1.7
1.7
22.9
24.0
0.6
0.8
Xanthine, 7-hydroxycoumarin
3.3.2. Further exploration of the enzymatic and non-enzymatic N-oxide
reduction of SNO in vitro
Each value represents the mean
SD of triplicates. N.D. = not detected.
- = not determined.
a
The metabolism of SNO was further verified in vitro. As shown in
Table 2, in the negative incubations without enzymes, SNO was stable
without the formation of SIN. However, the formation of SIN was ob-
served after SNO was incubated with HLMs or HLCs. To evaluate the
potential enzymatic and non-enzymatic metabolism of SNO, the roles of
xanthine oxidase, catalase, hemoglobin, and ferrous ion were further
assessed in the current study.
The incubation mixture (100 μL) was composed of SNO (10 μM) and
electron donor (NADH, NADPH and FAD) in potassium phosphate buffer
(100 mM, pH 7.4) in the absence of HLMs.
b
The incubation mixture (100 μL) was composed of SNO (10 μM) and HLMs
(1 mg/mL) in potassium phosphate buffer (100 mM, pH 7.4) in the absence of
electron donor (NADH, NADPH and FAD).
c
The incubation mixture (100 μL) was composed of SNO (10 μM) and
electron donor (NADH, NADPH and FAD) in potassium phosphate buffer
(100 mM, pH 7.4) in the absence of HLCs.
Non-enzymatic N-oxide reduction of SNO in HLMs (Table 2): SNO
was stable when it was incubated only with cofactors. However, after
the incubations of HLMs with SNO (without cofactors), the formation of
SIN was observed both in native and boiled HLMs without a significant
difference. After the addition of the electron donor (NADH and
NADPH), the reduction of SNO increased. Also, the increased N-oxide
reduction of SNO can be inhibited by sodium azide, an electron transfer
blocker. As is reported, heme moiety of hemoproteins can catalyze N-
oxide reduction, and the N-oxide reduction can be increased by the
addition of electron donors and inhibited by the addition of sodium
azide [28,29], suggesting that the N-oxide reduction of SNO in HLMs
was probably mediated by the heme moiety of hemoproteins in HLMs.
Enzymatic N-oxide reduction of SNO in HLCs (Table 2): SIN was
detected when SNO was incubated in HLCs with xanthine. Xanthine is
the electron donor of XOD. N-oxide reduction of SNO also occurred in
boiled HLCs, whereas the reduction activity in native HLCs was ex-
ceedingly higher in comparison to that in boiled HLCs. The addition of
7-hydroxycoumarin, a potent inhibitor of XOD, can inhibit the N-oxide
reduction of SNO, indicating that XOD was involved in the N-oxide
d
The incubation mixture (100 μL) was composed of SNO (10 μM) and HLCs
(1 mg/mL) in potassium phosphate buffer (100 mM, pH 7.4) in the absence of
electron donor (NADH, NADPH and FAD).
reduction of SNO.
Non-enzymatic N-oxide reduction of SNO in HLCs (Table 2): Both
native and boiled HLCs were able to mediate the N-oxide reduction of
SNO. The addition of NADPH can increase the metabolism of SNO. FAD
strikingly stimulated the N-oxide reduction of SNO in both native and
boiled HLCs. Yet, the N-oxide reductive activity was inhibited by so-
dium azide, inferring that heme moiety of hemoproteins in HLCs may
mediate the metabolism of SNO with the formation of SIN.
Non-enzymatic N-oxide reduction of SNO in denatured catalase,
denatured hemoglobin, and ferrous ion (Table 3): After the incubations
of SNO (10 μM) with boiled catalase for 120 min (without NADPH), the
average formation rate of SIN was 1.36
0.315 pmol/min/mg. When
NADPH was added into the incubation, the average formation rate
8

Q. Li, et al.
LifeSciences261(2020)118433
Table 3
production induced by LPS in Raw264.7 cells yet to promote the ROS
Non-enzymatic N-oxide reduction of SNO in boiled hemoglobin and ferrous ion.
production remarkably.
Addition
SIN formation (pmol/min/mg)
Boiled hemoglobin
N.D.
4. Discussion
Ferrous ion
Only a small amount of SIN, as reported, was found to be unchanged
after its oral administration [30], inferring plenty of metabolisms oc-
curred. Although a few studies reported the pharmacokinetics of SIN in
humans and rats, the major SIN's metabolites with the relevant meta-
bolic mechanism are still unclear. Only one research was done to
identify the metabolites of SIN in vivo. After the administration of SIN
to rats, three metabolites were reported and identified as 4-hydroxy-
3,7,7-trimethoxy-17-methyl-(9a,13a,14a)-morphinan-6-one (m/z at
None (negative incubation)a
None (negative)
NADPH
N.D.
N.D.c
N.D.
7.1
3.6b
4.1
2.1
43.0
92.6
NADH and FAD
8.4
0.9
Each value represents the mean
SD of triplicates. N.D. = not detected.
a
The incubation mixture (100 μL) was composed of SNO (10 μM) and
electron donor (NADH, NADPH and FAD) in potassium phosphate buffer
(100 mM, pH 7.4) in the absence of boiled hemoglobin and ferrous ion.
b
362),
7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl-N-oxide-
The incubation mixture (100 μL) was composed of SNO (10 μM) and boiled
(9a,13a,14a)-morphinan-6-one (m/z at 346, SNO), and 7,8-didehydro-
4-hydroxy-3,7-dimethoxy-(9a,13a,14a)-morphinan-6-one (m/z at 316,
DS) [18]. In our study, SNO and DS were found to be the most pre-
dominant metabolites of SIN both in humans and in rats for the first
time.
hemoglobin (1 mg/mL) in potassium phosphate buffer (100 mM, pH 7.4) in the
absence of electron donor (NADH, NADPH and FAD).
c
The incubation mixture (100 μL) was composed of SNO (10 μM) and fer-
rous ion (61.5 μM) in potassium phosphate buffer (100 mM, pH 7.4) in the
absence of electron donor (NADH, NADPH and FAD).
Although CYP3A4 and CYP2D6 were found to mediate the meta-
bolism of SIN using the substrate depletion method in another study,
the metabolites of SIN and the relevant metabolic mechanism were not
identified [14]. The current study provided evidence that CYP3A4 and
CYP2C19 primarily mediated the demethylation of SIN with the for-
mation of DS, and CYP3A4 primarily mediated the oxidation of SIN
with the formation of SNO in humans. CYP2D6*2 and CYP2D6*10 are
the most common genotypes of CYP2D6 in Asians [31]. CYP2D6*2 was
also proved to be involved in the demethylation of SIN, while it was not
the major CYP450s mediating the metabolism. The less formation of
SNO was observed in incubations of microsomes or cytosols with SIN in
the absence of NADPH and even in incubations of denatured enzymes
with SIN, indicating non-enzymatic oxidation of SIN. In our study, ROS
(H2O2 and O2-) was verified to be involved in the formation of SNO.
Species differences in the expression and activity of drug-metabolizing
enzymes exist, which limits the extrapolation from rats to humans.
Gender-related metabolism of drugs is often observed in rats, whereas
it's barely observed in other species [32]. Based on the results of
CYP450 enzyme kinetics, gender differences in the metabolism of SIN in
humans and rats were observed. However, the moderately higher ex-
pression and activity of CYP3A in male rats compared with female rats
[33] cannot explain the 13-fold higher Clint of SIN in MRLMs. The roles
of male-specific CYP450s (CYP2C11, CYP2C13 and CYP3A2) [34] on
SIN's metabolism in rats should not be ruled out. Especially, the es-
sential role of CYP2C11, compromising over 50% of total male hepatic
CYP450s [35], should be focused and further studied. In humans,
moderately higher CYP3A4 levels in females is proved [36], whereas
the significant variability of CYP3A4 expression, up to 50–100 fold
[37], should be more concerned than the limited gender differences.
The genetic polymorphisms of CYP2C19 can lead to variability in drug
responses. The risk of toxicity is frequently increased in CYP2C19 PMs
(poor metabolisers) [38,39]. Thus, the CYP2C19 PMs, IMs (inter-
mediate metabolisers) and EMs (extensive metabolisers) should be also
considered since these different phenotypes may lead to the different
levels of SIN's metabolism. Noteworthily, male rats may not be suitable
for SIN's pharmacodynamic and toxicological studies due to the ex-
ceptionally high Clint of SIN.
dramatically increased to 9.19
0.511 pmol/min/mg, inferring a
probable involvement of heme moiety of catalase in the N-oxide re-
duction of SNO. Also, hemoglobin, another kind of hemoproteins, was
used to verify the roles of heme moiety in SNO's metabolism. When SNO
was incubated with boiled hemoglobin (without cofactors), the for-
mation of SIN was observed. After the addition of the electron donor
(NADPH, NADH and FAD) to boiled hemoglobin, the N-oxide reductive
metabolism of SNO strikingly increased. Over 90% of SNO was con-
verted back to SIN in 120-min incubations of boiled hemoglobin with
SNO in the presence of NADH and FAD. Moreover, it was observed that
ferrous ion also limitedly mediated the metabolism of SNO in the pre-
sence of NADH and FAD. These facts suggested that ferrous ion and
heme moiety of hemoproteins played essential roles in the N-oxide
reduction of SNO.
3.4. Evidence of the anti-inflammation effects of SIN, DS and SNO and
oxidation property of SNO
3.4.1. Anti-inflammation effects of SIN, DS and SNO
In comparison to the control group (Fig. 6A, B and C), the levels of
IL-6 and TNF-α were remarkably increased in LPS-induced Raw264.7
cells and significantly attenuated by the addition of DEX. Pretreatment
of the cells with SIN (10, 100 and 200 μM) can exceedingly decrease the
production of IL-6 induced by LPS (Fig. 6A). Additionally, pretreatment
of the cells with DS and SNO (200 μM) can mitigate both IL-6 and TNF-
α levels in LPS-induced Raw264.7 cells (Fig. 6A and B). The effects of
DS and SNO (200 μM) on the TNF-α protein levels were further verified
by its mRNA levels (Fig. 6C). For the further mechanism study, the
impacts of SIN, DS and SNO on NF-κB signaling pathways were eval-
uated (Fig. 6D). The nuclear translocation of p-p65 protein in
RAW264.7 cells was remarkably increased after the treatment of LPS,
while DEX suppressed the translocation. In comparison to the LPS
group, SIN strikingly inhibited the nuclear translocation of p-p65 pro-
tein in a concentration-dependent manner. Pretreatment of the cells
with SNO at 10 and 100 μM was not able to suppress the nuclear
translocation of p-p65. All the observations suggested that, in com-
parison to DS and SNO, SIN played the more essential role in the anti-
inflammation effects in the LPS-induced Raw264.7 cells, while DS and
SNO, only at very high concentrations (far beyond the concentration in
vivo), have limited anti-inflammation effects.
To reveal the potential metabolic mechanism of SIN in a cyclic
fashion, the metabolism of DS and SNO was further evaluated in our
study. It was observed that DS was stable in vivo in rats, whereas SNO
can be metabolized into SIN with a trace formation of DS. Tertiary
amine N-oxides were mostly enzymatically reduced to its corresponding
amines by CYP450s [40]. However, non-enzymatic N-oxide reductive
metabolism was still observed in some tertiary amines [29,41]. The
abnormal N-oxide metabolism of amines was non-enzymatic metabo-
lism mediated by the heme moiety of hemoproteins and independent of
the enzymatic activity of the apoprotein, and it can be strikingly
3.4.2. Oxidation property of SNO
As shown in Fig. 6E, LPS significantly induced ROS production in
the LPS-induced Raw264.7 cells. After the pretreatment of H2O2, the
ROS production strikingly increased in comparison to the control and
the LPS group. Noteworthily, SNO was not able to attenuate the ROS
9

Q. Li, et al.
LifeSciences261(2020)118433
(caption on next page)
10

Q. Li, et al.
LifeSciences261(2020)118433
Fig. 6. Effects of SIN, SNO and DS on inflammatory response and impacts of SNO on ROS production in LPS-induced Raw264.7 cells. A. Protein levels of IL-6 in LPS-
induced Raw264.7 cells after pretreatment of SIN, SNO and DS (10, 100 and 200 μM). B. Protein levels of TNF-α in LPS-induced Raw264.7 cells after pretreatment of
SIN, SNO and DS (10, 100 and 200 μM). C. Changes in mRNA levels of TNF-α in LPS-induced Raw264.7 cells after pretreatment of SNO and DS (200 μM). D. Relative
intensity of nuclear translocation of NF-κB (p-p65/p65) in LPS-induced Raw264.7 cells after pretreatment of SIN, SNO and DS (10, 100 and 200 μM). E. Relative ROS
production in LPS-induced Raw264.7 cells after pretreatment of SNO (10 μM). Each data point is expressed as mean
SD of triplicates. *p < 0.05, **p < 0.01,
***p < 0.001, significant difference vs. the LPS group.
Fig. 7. Proposed schematic diagram of cyclic metabolism of SIN in humans.
promoted by electron donors and remarkably inhibited by sodium azide
[28,29,42]. In the current study, different kinds of hemoproteins were
boiled to make the apoproteins denatured, and yet plenty of SNO was
still converted to SIN, especially in the presence of electron donors,
suggesting that heme moiety of hemoproteins contributed to the non-
enzymatic N-oxide reduction of SNO. CYP450 isozymes do not seem to
be involved in the enzymatic reduction of SNO since the extent of re-
duction was identical in incubations of denatured isozymes or native
isozymes with SNO (data not shown), inferring the heme moiety of
CYP450s may mediate the metabolism of SNO instead. Also, ferrous ion
was testified to be involved in the metabolism of SNO. Besides, the
current study provided evidence that XOD, a cytosolic enzyme with N-
oxide reductive activities [42], directly participated in the enzymatic N-
oxide metabolism of SNO. Intriguingly, XOD also indirectly mediated
the formation of SNO, since XOD can lead to the formation of ROS and
subsequently mediated the metabolism of SIN with the formation of
SNO. In summary (Fig. 7), SIN predominantly underwent demethyla-
tion with the formation of DS by CYP3A4 and CYP2C19 and underwent
N-oxidation with the formation of SNO both enzymatically by CYP3A4
and non-enzymatically by ROS. Additionally, both enzymes (XOD) and
non-enzymes (ferrous ion and heme moiety of hemoproteins) mediated
SNO's metabolism with the formation of SIN in a cyclic fashion.
SIN underwent active hepatobiliary elimination mediated by P-
glycoprotein (P-gp) in rats [43], leading to an increased concern on
DDI. The bioavailability of paeoniflorin was enhanced by SIN since SIN
worked as a P-gp inhibitor and decreased the efflux transport of
paeoniflorin [13]. SIN also modulated the enzyme activity of CYP2C19
[44], inferring a possibility of DDI between SIN and CYP2C19 sub-
strates. To reduce the arthritis-related risk of cardiovascular diseases,
SIN was usually co-administered with simvastatin or lovastatin,
whereas long-term co-administration of simvastatin with SIN decreased
the systemic exposure of SIN in vivo [14]. The co-administration of SIN
with other drugs warrants more clinical focus. The current study pro-
vided firm evidence that enzymes (CYP450s and XOD) and non-en-
zymes (ROS, ferrous ion and heme moiety) mediated the cyclic meta-
bolism of SIN, which would be much helpful for further studies on DDI
when SIN was co-administrated with other drugs.
The anti-inflammation activity of SIN, as reported, is related to the
inhibition of NADPH oxidase and decrease of TNF-a, IL-1, ROS, NO and
PGE2 [45,46]. NF-κB is retained in the cytoplasm, and the nuclear
translocation of NF-κB simultaneously regulates plenty of cytokines
including TNF-α, IL-1β and IL-6 [47]. These cytokines lead to in-
flammatory responses [48]. The current study revealed that SIN, SNO
and DS were able to attenuate the inflammation induced by LPS in the
Raw264.7 cells. The levels of cytokines and nuclear translocation of NF-
κB were remarkably suppressed by the pretreatment of SIN in a con-
centration-dependent manner, while SNO and DS (200 μM) only
slightly attenuated these in LPS-induced Raw264.7 cells. The Cmax of
SIN after its subcutaneous administration (9 mg/kg) in rats is about
4 μM [49], and that after oral administration of SIN tablets/capsules
(120 mg/day) in dogs and humans is 1 μM to 5 μM [15,50]. DS and SNO
alleviated inflammation only at extremely high concentrations, in-
dicating that they may not play the major anti-inflammatory roles in
vivo when conventional regimen of SIN is administrated to RA patients.
Thus, SIN was revealed as the most predominant compound mediating
the anti-inflammation effects in comparison to SNO and DS, inferring
that intra-articular injection may be more recommended to eliminate
the inert metabolism of SIN and improve its therapeutic effects. Note-
worthily, DS was reported as a more potent immunosuppressive com-
pound in comparison to SIN. DS suppressed IL-2 with IC50 of 20 μM
11

Q. Li, et al.
LifeSciences261(2020)118433
[17]. Therefore, further researches on DS may be necessary.
References
Side effects, including mild pain, dyspnea and diarrhea are fre-
quently observed after the administration of SIN [51]. The potential
toxicities of SIN and its major metabolites should be considered. It is
noteworthy that hemoproteins, including hemoglobin, myoglobin,
catalase, peroxidase, cytochrome c and cytochrome b5, play the essen-
tial roles in cellular energy homeostasis and charge for the transport of
oxygen and redox metabolism of endogenous and exogenous com-
pounds. The current study elucidated that SNO can react with ferrous
ion and heme moiety of hemoproteins. Moreover, SNO at 10 μM cannot
attenuate cytokines and nuclear translocation of NF-κB in LPS-induced
Raw264.7 cells, whereas it significantly induced ROS formation. ROS
mostly leads to pathological processes [52–54]. The toxic effects of ROS
include lipid peroxidation of the membrane [55] and damage of pro-
teins and nucleic acids [56,57]. The current study elucidated that
CYP3A4 mediated the metabolism of SIN with the formation of SNO at
a high rate, leading to the considerable formation of SNO in humans
after oral administration of the SIN-containing drugs. SNO can further
induce the ROS formation, inferring a potential oxidative stress and
damage to the organism. Besides, since hemeproteins widely locate in
the body, the continuous cyclic metabolism of SIN may wildly occur
until SIN and its metabolites are eliminated. The interrelated amount of
SIN, SNO and DS in vivo may affect the pharmacological effects and
may lead to side effects or even toxic effects. To warrant the therapeutic
effects and clinical safety of SIN, more researches on SIN with a concern
of SNO and DS are needed.
[1] Chinese Pharmacopoeia Commission, Chinese Pharmacopoeia, 2015 ed., Chemical
Industry Press, Beijing, 2015.
[2] D.L. Scott, D.P. Symmons, B.L. Coulton, A.J. Popert, Long-term outcome of treating
rheumatoid arthritis: results after 20 years, Lancet 1 (1987) 1108–1111, https://
doi.org/10.1016/s0140-6736(87)91672-2.
[3] D.L. Scott, F. Wolfe, T.W. Huizinga, Rheumatoid arthritis, Lancet 376 (2010)
1094–1108, https://doi.org/10.1016/S0140-6736(10)60826-4.
[4] X.H. Wang, S.H. Qiu, S.X. Dong, P. Qiu, F.C. Wu, Studies on the pharmacodynamics
of Sinomenine tablets, Pharmacol. Clin. Chinese Mater. Med. 13 (1997) 23–25,
https://doi.org/10.13412/j.cnki. zyyl.1997.04.011.
[5] M. Xu, L. Liu, C. Qi, B. Deng, X. Cai, Sinomenine versus NSAIDs for the treatment of
rheumatoid arthritis: a systematic review and meta-analysis, Planta Med. 74 (2008)
1423–1429, https://doi.org/10.1055/s-2008-1081346.
[6] Y.X. Liu, C.P. Zhao, Q.H. Ma, Y. Li, Sinomenine retards LPS-elicited inflammation
via down-regulating CCAT1 in HaCaT cells, Life Sci. 233 (2019) 1–9, https://doi.
org/10.1016/j.lfs.2019.116703.
[7] D.P. Chen, C.K. Wong, P.C. Leung, K.P. Fung, C.B. Lau, C.P. Lau, E.K. Li, L.S. Tam,
C.W. Lam, Anti-inflammatory activities of Chinese herbal medicine sinomenine and
Liang Miao San on tumor necrosis factor-α-activated human fibroblast-like syno-
viocytes in rheumatoid arthritis, J. Ethnopharmacol. 137 (2011) 457–468, https://
doi.org/10.1016/j.jep.2011.05.048.
[8] G. Nagy, J.M. Clark, E.I. Buzas´, C.L. Gorman, A.P. Cope, Nitric oxide, chronic in-
flammation and autoimmunity, Immunol. Lett. 111 (2007) 1–5, https://doi.org/10.
1016/j.imlet.2007.04.013.
[9] C.F. Nathan, Neutrophil activation on biological surfaces. Massive secretion of
hydrogen peroxide in response to products of macrophages and lymphocytes, J.
Clin. Invest. 80 (1987) 1550–1560, https://doi.org/10.1172/jci113241.
[10] G.J. Wu, T.L. Chen, Y.F. Ueng, R.M. Chen, Ketamine inhibits tumor necrosis factor-
alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macro-
phages through suppression of toll-like receptor 4-mediated c-Jun N-terminal ki-
nase phosphorylation and activator protein-1 activation, Toxicol. Appl. Pharmacol.
228 (2008) 105–113, https://doi.org/10.1016/j.taap.2007.11.027.
[11] L. Yi, Y.J. Lyn, C. Peng, R.L. Zhu, S.S. Bai, L. Liu, P.X. Wang, H. Zhou, Y. Dong,
Sinomenine inhibits fibroblast-like synoviocyte proliferation by regulating
α7nAChR expression via ERK/Egr-1 pathway, Int. Immunopharmacol. 56 (2018)
65–70, https://doi.org/10.1016/j.intimp. 2018.01.015.
5. Conclusion
[12] P. Teng, H.L. Liu, L. Zhang, L.L. Feng, Y. Huai, Z.S. Deng, Y. Sun, Q. Xu, J.X. Li,
Synthesis and biological evaluation of novel sinomenine derivatives as anti-in-
flammatory agents, Eur. J. Med. Chem. 50 (2012) 63–74, https://doi.org/10.1016/
j.ejmech. 2012.01.036.
In conclusion, SIN was predominately metabolized to SNO by
CYP3A4 and ROS (H2O2 and O2-) and to DS by CYP3A4 and CYP2C19.
SNO underwent N-oxide reduction both enzymatically, by XOD, and
non-enzymatically, by ferrous ion and heme moiety of hemoproteins
including catalase, hemoglobin and CYP450s, while DS was stable.
Additionally, SIN played the primary anti-inflammation role in com-
parison to its major metabolites SNO and DS in LPS-induced Raw264.7
cells. SNO (10 μM) was not able to attenuate the inflammation re-
sponses, while it can conversely induce ROS production in LPS-induced
Raw264.7 cells.
[13] K. Chan, Z.Q. Liu, Z.H. Jiang, H. Zhou, Y.F. Wong, H.X. Xu, L. Liu, The effects of
sinomenine on intestinal absorption of paeoniflorin by the everted rat gut sac
model, J. Ethnopharmacol. 103 (2006) 425–432, https://doi.org/10.1016/j.jep.
2005.08.020.
[14] Y. Wang, Y. Jin, X.M. Yun, M.J. Wang, Y. Dai, Y.F. Xia, Co-administration with
simvastatin or lovastatin alters the pharmacokinetic profile of sinomenine in rats
through cytochrome P450-mediated pathways, Life Sci. 209 (2018) 228–235,
https://doi.org/10.1016/j.lfs.2018.08.012.
[15] J. Sun, J.M. Shi, T.H. Zhang, K. Gao, J.J. Mao, B. Li, Y.H. Sun, Z.G. He, Impact of
release characteristics of sinomenine hydrochloride dosage forms on its pharma-
cokinetics in beagle dogs, World J. Gastroenterol. 11 (2005) 4547–4551, https://
doi.org/10.3748/wjg. v11.i29.4547.
CRedit authorship contribution statement
[16] Y.M. Yao, Z.R. Tan, Z.Y. Hu, X. Guo, Z.N. Cheng, L.S. Wang, H.H. Zhou,
Determination of sinomenine in human plasma by HPLC/ESI/ion trap mass spec-
trum, Clin. Chim. Acta 356 (2005) 212–217, https://doi.org/10.1016/j.cccn.2005.
01.018.
Qiang Li: Investigation, Resources, Formal analysis, Writing-ori-
ginal Draft. Wenbin Zhou: Validation, Formal analysis, Writing-
Review & Editing. Yuyan Wang: Validation, Writing-Review & Editing.
Fang Kou: Resources. Chunming Lyu: Conceptualization,
Methodology, Formal analysis, Investigation, Writing-original Draft,
Writing-Review & Editing, Supervision. Hai Wei: Conceptualization,
Writing-Review & Editing, Supervision, Resources.
[17] U. Christians, V.W. Kaever, Epimorphian compound and its use, World Intellectual
Property Organization Publ. of the Int. Appl. With Int. Search Report
WO1999US03753, Canada, 1999.
[18] W.M. Cheng, F. Qiu, X.S. Yao, Three major urinary metabolites of sinomenine in
rats, J. Asian Nat. Prod. Res. 9 (2007) 13–18, https://doi.org/10.1080/
10286020500289444.
[19] Z.L. Zhang, Q. Li, Q.F. Li, S.M. Du, Y.Q. Zhou, C.M. Lv, Y. Zhao, Y.J. Wang,
N. Zhang, Simultaneous determination of nineteen major components in Qi She Pill
by ultra-high-performance liquid chromatography-tandem mass spectrometry, Acta
Pharm. Sin. B 4 (2014) 384–393, https://doi.org/10.1016/j.apsb.2014.05.003.
[20] Y.L. Sun, T. Hou, S.F. Liu, Z.L. Zhang, N. Zhang, M. Yao, L. Yang, Q. Shi, X.J. Cui,
Y.J. Wang, Population pharmacokinetic modeling of the Qishe pill in three major
traditional Chinese medicine-defined constitutional types of healthy Chinese sub-
jects: study protocol for a randomized controlled trial, Trials 16 (2015) 1–10,
https://doi.org/10.1186/s13063-015-0568-6.
Declaration of competing interest
The authors declare that there are no potential conflicts of interest.
[21] C.M. Lyu, Y.F. Zhang, W.B. Zhou, S. Zhang, F. Kou, H. Wei, N. Zhang, Z. Zuo,
Gender-dependent pharmacokinetics of veratramine in rats: in vivo and in vitro
evidence, AAPS J. 18 (2016) 432–444, https://doi.org/10.1208/s12248-016-
9870-9.
Acknowledgments
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
[22] D.A. Kostić, D.S. Dimitrijević, G.S. Stojanović, I.R. Palić, A.S. Đorđević,
J.D. Ickovski, Xanthine oxidase: isolation, assays of activity, and inhibition, J.
Chem. 2015 (2015) 1–8, https://doi.org/10.1155/2015/294858.
[23] E.M. Link, P.A. Riley, Role of hydrogen peroxide in the cytotoxicity of the xanthine/
xanthine oxidase system, Biochem. J. 249 (1988) 391–399, https://doi.org/10.
1042/bj2490391.
Appendix A. Supplementary data
[24] J.M. Hutzler, T.S. Tracy, Atypical kinetic profiles in drug metabolism reactions,
Drug Metab. Dispos. 30 (2002) 355–362, https://doi.org/10.1124/dmd.30.4.355.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.lfs.2020.118433.
12

Q. Li, et al.
LifeSciences261(2020)118433
[25] W.S. Chang, H.C. Chiang, Structure-activity relationship of coumarins in xanthine
oxidase inhibition, Anticancer Res. 15 (1995) 1969–1973 (PMID: 8572586).
[26] S.E. Choi, S.H. Min, H.C. Shin, H.E. Kim, M.W. Jung, Y. Kang, Involvement of
calcium-mediated apoptotic signals in H2O2-induced MIN6N8a cell death, Eur. J.
Pharmacol. 547 (2006) 1–9, https://doi.org/10.1016/j.ejphar.2006.06.016.
[27] A.I. Cederbaum, Oxygen radical generation by microsomes: role of iron and im-
plications for alcohol metabolism and toxicity, Free Radic. Biol. Med. 7 (1989)
559–567, https://doi.org/10.1016/0891-5849(89)90033-6.
Metab. Dispos. 27 (1999) 92–97.
[42] M. Zheng, J. Jiang, J.P. Wang, X.Q. Tang, M. Ouyang, Y.Q. Deng, The mechanism of
enzymatic and non-enzymatic N-oxide reductive metabolism of cyadox in pig liver,
Xenobiotica 41 (2011) 964–971, https://doi.org/10.3109/00498254.2011.593207.
[43] T.H. Tsai, J.W. Wu, Regulation of hepatobiliary excretion of sinomenine by P-gly-
coprotein in Sprague-Dawley rats, Life Sci. 72 (2003) 2413–2426, https://doi.org/
10.1016/s0024-3205(03)00118-8.
[44] Y.M. Yao, W. Cao, Y.J. Cao, Z.N. Cheng, D.S. Ouyang, Z.Q. Liu, H.H. Zhou, Effect of
sinomenine on human cytochrome P450 activity, Clin. Chim. Acta 379 (2007)
113–118, https://doi.org/10.1016/j.cca.2006.12.022.
[28] K. Takekawa, S. Kitamura, K. Sugihara, S. Ohta, Non-enzymatic reduction of ali-
phatic tertiary amine N-oxides mediated by the haem moiety of cytochrome P450,
Xenobiotica 31 (2001) 11–23, https://doi.org/10.1080/00498250010024997.
[29] K. Takekawa, K. Sugihara, S. Kitamura, K. Tatsumi, Nonenzymatic reduction of
brucine N-oxide by the heme group of cytochrome P450, Biochem. Mol. Biol. Int. 42
(1997) 977–981, https://doi.org/10.1080/15216549700203421.
[45] L. Qian, Z.L. Xu, W. Zhang, B. Wilson, J.S. Hong, P.M. Flood, Sinomenine, a natural
dextrorotatory morphinan analog, is anti-inflammatory and neuroprotective
through inhibition of microglial NADPH oxidase, J. Neuroinflammation 4 (2007)
1–14, https://doi.org/10.1186/1742-2094-4-23.
[30] X.H. Yan, H.D. Li, W.X. Peng, F.Q. Liu, Y. Shao, Y.Q. He, Determination of sino-
menine HCl in serum and urine by HPLC and its pharmacokinetics in normal vo-
lunteers, Acta Pharm. Sin. (1997) 620–624, https://doi.org/10.16438/j.0513-4870.
1997.08.014.
[46] Y. Wang, Y.F. Fang, W.H. Huang, X. Zhou, M.H. Wang, B. Zhong, D.Z. Peng, Effect
of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant
arthritis rats, J. Ethnopharmacol. 98 (2005) 37–43, https://doi.org/10.1016/j. jep.
2004. 12.022.
[31] M.J. Kim, J.Y. Byeon, Y.H. Kim, S.H. Kim, C.M. Lee, E.H. Jung, W.K. Chae, Y.J. Lee,
C.G. Jang, S.Y. Lee, C.I. Choi, Effect of the CYP2D6*10 allele on the pharmacoki-
netics of clomiphene and its active metabolites, Arch. Pharm. Res. 41 (2018)
347–353, https://doi.org/10.1007/s12272-018-1005-7.
[47] T.T. Vuong, S.B. Rønning, H.P. Suso, R. Schmidt, K. Prydz, M. Lundström, A. Moen,
M.E. Pedersen, The extracellular matrix of eggshell displays anti-inflammatory ac-
tivities through NF-κB in LPS-triggered human immune cells, J. Inflamm. Res. 10
(2017) 83–96, https://doi.org/10.2147/jir.s130974.
[32] R. Kato, Y. Yamazoe, Sex-specific cytochrome P450 as a cause of sex- and species-
related differences in drug toxicity, Toxicol. Lett. 64-65 (1992) 661–667, https://
doi.org/10.1016/0378-4274(92)90245-f.
[48] H.R. Li, J. Liu, S.L. Zhang, T. Luo, F. Wu, J.H. Dong, Y.J. Guo, L. Zhao, Corilagin
ameliorates the extreme inflammatory status in sepsis through TLR4 signaling
pathways, BMC Complement. Med. Ther. 17 (2017) 1–8, https://doi.org/10.1186/
s12906-016-1533-y.
[33] P. P. Souček, I. Gut, Cytochromes P-450 in rats: structures, functions, properties and
relevant human forms, Xenobiotica 22 (1992) 83–103, https://doi.org/10.3109/
00498259209053106.
[49] Y.C. XIE, Q.F. ZHU, Z.Y. FAN, M. GUO, Determination of sinomenine HCl in serum
by HPLC and its pharmacokinetics after different administration, Chin. Pharm. J. 40
(2005) 54–55.
[34] C. Thangavel, W. Dworakowski, B.H. Shapiro, Inducibility of male-specific isoforms
of cytochrome p450 by sex-dependent growth hormone profiles in hepatocyte
cultures from male but not female rats, Drug Metab. Dispos. 34 (2006) 410–419,
https://doi.org/10.1124/dmd.105.007716.
[50] M.X. Su, M. Song, D.Z. Sun, H. Zhao, X. Gu, L. Zhu, X.L. Zhan, Z.N. Xu, A.D. Wen,
T.J. Hang, Determination of sinomenine sustained-release capsules in healthy
Chinese volunteers by liquid chromatography-tandem mass spectrometry, J.
Chromatogr. B Analyt. Technol. Biomed. Life Sci. 889–890 (2012) 39–43, https://
doi.org/10.1016/j.jchromb.2012.01.027.
[35] E.T. Morgan, C. MacGeoch, J.A. Gustafsson, Hormonal and developmental regula-
tion of expression of the hepatic microsomal steroid 16 alpha-hydroxylase cyto-
chrome P-450 apoprotein in the rat, J. Biol. Chem. 260 (1985) 11895–11898.
[36] R. Wolbold, K. Klein, O. Burk, A.K. Nüssler, P. Neuhaus, M. Eichelbaum, M. Schwab,
U.M. Zanger, Sex is a major determinant of CYP3A4 expression in human liver,
Hepatology 38 (2003) 978–988, https://doi.org/10.1053/jhep.2003.50393.
[37] A. Westlind, L. Löfberg, N. Tindberg, T.B. Andersson, M. Ingelman-Sundberg,
Interindividual differences in hepatic expression of CYP3A4: relationship to genetic
polymorphism in the 5′-upstream regulatory region, Biochem. Biophys. Res.
Commun. 259 (1999) 201–205, https://doi.org/10.1006/bbrc.1999.0752.
[38] K.I. Lorenzini, A. Calmy, J. Ambrosioni, B. Assouline, Y. Daali, M. Fathi,
M. Rebsamen, J. Desmeules, C.F. Samer, Serotonin syndrome following drug-drug
interactions and CYP2D6 and CYP2C19 genetic polymorphisms in an HIV-infected
patient, AIDS 26 (2012) 2417–2418, https://doi.org/10.1097/QAD.
0b013e32835a11ba.
[51] Z.Q. Liu, K. Chan, H. Zhou, Z.H. Jiang, Y.F. Wong, H.X. Xu, L. Liu, The pharma-
cokinetics and tissue distribution of sinomenine in rats and its protein binding
ability in vitro, Life Sci. 77 (2005) 3197–3209, https://doi.org/10.1016/j.lfs.2005.
05.054.
[52] M.T. Lin, M.F. Beal, Mitochondrial dysfunction and oxidative stress in neurode-
generative diseases, Nature 443 (2006) 787–795, https://doi.org/10.1038/nature
05292.
[53] A.P. Wagner, S. Mitran, S. Sivanesan, E. Chang, A.M. Buga, ROS and brain diseases:
the good, the bad, and the ugly, Oxidative Med. Cell. Longev. (2013) 1–14, https://
doi.org/10.1155/2013/963520.
[54] G.H. Kim, E. Jieun, J.E. Kim, S.J. Rhie, S.J. Yoon, The role of oxidative stress in
neurodegenerative diseases, Exp. Neurobiol. 24 (2015) 325–340, https://doi.org/
10.5607/en.2015.24.4.325.
−
[39] S.F. Zhou, J.P. Liu, B. Chowbay, Polymorphism of human cytochrome P450 en-
zymes and its clinical impact, Drug Metab. Rev. 41 (2009) 89–295, https://doi.org/
10.1080/03602530902843483.
[55] B.H.J. Bielski, R.L. Arudi, M.W. Sutherland, A study of the reactivity of HO2/O2
with unsaturated fatty acids, J. Biol. Chem. 258 (1983) 4759–4761.
[56] J.M. Gutteridge, S. Wilkins, Copper salt-dependent hydroxyl radical formation.
Damage to proteins acting as antioxidants, Biochim. Biophys. Acta 759 (1983)
38–41, https://doi.org/10.1016/0304-4165(83)90186-1.
[40] M. Sugiura, R. Iwasaki, H. Noguchi, R. Kato, Evidence for the involvement of cy-
tochrome P-450 in tiaramide N-oxide reduction, Life Sci. 15 (1974) 1433–1442,
https://doi.org/10.1016/0024-3205(74)90118-0.
[57] H.R. Massie, H.V. Samis, M.B. Baird, The kinetics of degradation of DNA and RNA
by H2O2, Biochim. Biophys. Acta 272 (1972) 539–548, https://doi.org/10.1016/
0005-2787(72)90509-6.
[41] S. Kitamura, K. Sugihara, K. Tatsumi, A unique tertiary amine N-oxide reduction
system composed of quinone reductase and heme in rat liver preparations, Drug
13