Structural elucidation of in vitro metabolites of bavachinin in rat liver microsomes by LC-ESI-MSn and chemical synthesis

Article abstract of DOI:10.3109/00498254.2015.1074763

1. Bavachinin isolated from Psoralea corylifolia has various activities, such as antimicrobial, antiallergic, antitumor and so on. Our previous study showed that natural bavachinin exhibits peroxisome proliferator-activated receptor γ-agonist activity.2.

Full text of DOI:10.3109/00498254.2015.1074763

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Structural elucidation of in vitro metabolites of
bavachinin in rat liver microsomes by LC-ESI-MSn and
chemical synthesis
Fan Xiea, Guoxin Dua, Shunan Maa, Yiming Lia, Rui Wanga & Fujiang Guoa
a School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, P. R.
China
Published online: 14 Aug 2015.
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To cite this article: Fan Xie, Guoxin Du, Shunan Ma, Yiming Li, Rui Wang & Fujiang Guo (2015): Structural elucidation of in
vitro metabolites of bavachinin in rat liver microsomes by LC-ESI-MSn and chemical synthesis, Xenobiotica: the fate of foreign
compounds in biological systems
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2015 Taylor & Francis. DOI: 10.3109/00498254.2015.1074763
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RESEARCH ARTICLE
Structural elucidation of in vitro metabolites of bavachinin in rat liver
microsomes by LC-ESI-MSn and chemical synthesis
Fan Xie, Guoxin Du, Shunan Ma, Yiming Li, Rui Wang, and Fujiang Guo
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, P. R. China
Abstract
Keywords
1. Bavachinin isolated from Psoralea corylifolia has various activities, such as antimicrobial,
antiallergic, antitumor and so on. Our previous study showed that natural bavachinin
exhibits peroxisome proliferator-activated receptor g-agonist activity.
2. In vitro studies on bavachinin metabolism were conducted using rat liver microsomes
incubated at 37 ꢀC for 60 min.
3. Structures of eight metabolites of the incubation mixtures were cautiously characterized
using electrospray tandem mass spectra and three synthetic compounds. The results
indicated that eight metabolites of bavachinin were biotransformed mainly through
oxidation.
Bavachinin, chemical synthesis, in vitro
metabolism, mass spectrometry, rat liver
microsomes
History
Received 17 June 2015
Revised 15 July 2015
Accepted 16 July 2015
Published online 10 August 2015
4. The metabolic pathways of bavachinin were elucidated in vitro. These results contribute to
the understanding of bavachinin’s in vivo metabolism.
Introduction
nuclear hormone receptors’ superfamily and are essential in
the regulation of cellular differentiation, development, metab-
olism and tumorigenesis of higher organisms (Singh et al.,
2011). PPAR-g is a key regulator of insulin sensitization and
glucose metabolism (Chigurupati et al., 2015), and PPAR-g
activation is extremely effective in improving glycemic
management; however, little is known about the metabolism
of bavachinin.
Dry and ripe seeds of leguminous plant Psoralea corylifolia
L. (bu-gu-zhi in Chinese) have been widely used for treating
asthma, diarrhea, waist and knee psychroalgia (Luo et al.,
2014), osteoporosis (Huang et al., 2014; Li et al., 2014),
kidney insufficiency, enuresis, vitiligo and alopecia areata
(Pharmacopeia Committee of P.R. China, 2010). Most of its
chemical constituents, including flavonoids, furanocoumarins
and monoterpene phenols, have been isolated, a few of which
exhibit antioxidant (Guo et al., 2005), antimicrobial and
hepatoprotective activities (Chopra et al., 2013); DNA
polymerase and topoisomerase II inhibition (Sun et al.,
1998); and antidermatophytic (Lau et al., 2014), antiallergic
(Matsuda et al., 2007) and antitumor (Chen et al., 2010)
activities.
In vitro metabolic studies are gaining increasing attention
for several reasons, including their speediness, simplicity,
convenience and low cost. Moreover, in vitro metabolism
studies can eliminate the disturbance of factors in vivo to
observe the selective metabolism of metabolic enzyme on
substrate, which will get more reliable theory basis for in vivo
metabolism. Methods used to study the in vitro metabolism
studies of flavonoids include the Caco-2 cell model, gastro-
intestinal contents hatching, the enterohepatic microsomal
method, and the genetic recombination metabolic enzyme
method (Aura et al., 2005; Boersma et al., 2002; Chen et al.,
2005; Si et al., 2009). Except these, liver microsomes play an
important role in researching the metabolism of flavonoids,
because the liver is the crucial metabolism site (Wang & Ho,
2009). Several studies on the metabolism of flavonoids in
liver microsomes have been reported (Lee et al., 2007;
Nikolic et al., 2005; Quintieri et al., 2008).
Bavachinin (Figure 1), isolated from P. corylifolia (Chen
et al., 2012), is a prenylated flavonoid. Bavachinin exhibits
antiallergic, anti-inflammatory (Chen et al., 2013, 2014),
antiangiogenic, antitumor (Nepal et al., 2012) and antioxidant
activities (Xiao et al., 2010), and inhibits severe acute
respiratory syndrome coronavirus papain-like protease (Kim
et al., 2014). In our study (WO2014/169800), natural
bavachinin exhibited peroxisome proliferator-activated recep-
tor g (PPAR-g) agonist activity. PPARs are members of the
The aim of this study is to identify the metabolites of
bavachinin in rat liver microsomes and to speculate the
metabolic pathways of bavachinin by mass spectrometry
combined with liquid chromatography and chemical
synthesis.
Address for correspondence: Prof. Fujiang Guo and Prof. Rui Wang,
School of Pharmacy, Shanghai University of Traditional Chinese
Medicine, 1200 Cailun Road, Shanghai, 201203, P. R. China. Tel: +
86 21 51322181. Fax: + 86 21 51322193. E-mail: gfj@shutcm.edu.cn
(F. Guo); ellewang@163.com (R. Wang).

2
F. Xie et al.
Xenobiotica, Early Online: 1–11
(20 mL) at room temperature and stirred for 2 h (Scheme 2).
The reaction mixture was monitored using TLC. The reaction
mixture was quenched using water (40 mL) and extracted
using ethyl acetate (2 ꢁ 20 mL). The organic layers were
combined, washed using a saturated NaCl solution (20 mL),
dried over Na2SO4, filtrated and evaporated in vacuum. The
residue was purified by silica gel column chromatography
with petroleum ether/ethyl acetate to yield a colorless oil, S2b
(646 mg, 78.88%). Potassium trimethylsilanolate (1320 mg,
9.19 mM) was added to a stirred solution of S2c (652 mg,
2.78 mM) and S2b (630 mg, 2.78 mM) in ethanol (20 mL),
and the mixture was refluxed in an inert atmosphere for 4 h.
The reaction mixture was monitored using TLC. The reaction
mixture was quenched using saturated aqueous NH4Cl
(20 mL) and extracted using ethyl acetate (2 ꢁ 20 mL). The
organic layers were processed as previously described to yield
S2d as bright yellow needles (460.3 mg, 37.38%). Potassium
fluoride (230 mg, 3.96 mM) was added to a solution of S2d
(460.3 mg, 1.04 mM) in methanol (10 mL). The mixture was
refluxed with stirring for 8 h. The reaction mixture was
monitored using TLC. The reaction mixture was quenched
and extracted using water (10 mL) and ethyl acetate
(2 ꢁ 10 mL), respectively. The organic layers were processed
as previously described to yield S2e as a white solid
(269.3 mg, 58.51%). Aqueous hydrochloric acid (3 N,
2.26 mL) was added to a solution of S2e (200 mg, 0.45 mM)
in methanol (7 mL), and the mixture was refluxed for 10 min.
After cooling and diluting with water (10 mL), the mixture
was extracted using ethyl acetate (2 ꢁ 10 mL). The organic
layers were processed as previously described to yield S2f as a
Figure 1. Molecular structure of bavachinin.
Materials and methods
Chemicals
Bavachinin was isolated from P. corylifolia L as previously
described (Yin et al., 2004); its purity was determined to be
>98% by using a high-performance liquid chromatography
(HPLC) analysis (Hyper 0DS2 C18 column; 4.6 mm ꢁ 250
mm, 5 mm). Separation was performed under the following
conditions: flow rate, 1.0 mL/min; solvent A, 0.3% phos-
phoric acid in water; solvent B, CH3CN; 0–15 min, 50–95%
B; 15–20 min, 95–95% B; 20–25 min, 95–50% B (v/v).
HPLC-grade solvents were purchased from Fisher Scientific
Co. (Fair Lawn, NJ). All other chemicals were purchased
from Sinopharm Chemical Reagent Co. (Shanghai, China).
Synthesis of 4’-hydroxy-7-methoxy-6 -(3-methyl-2-
butenyl)-flavone (S1b)
1
pale yellow solid (96.5 mg, 60.24%). H NMR ꢀ (400 MHz,
Iodine (94 mg, 0.74 mM) was added to a solution of
bavachinin (S1a) (250 mg, 0.74 mM) in DMSO (10 mL).
The mixture was heated to 90 ꢀC and stirred for 3 h. The
reaction mixture was monitored through thin layer chroma-
tography (TLC). After cooling, the reaction mixture was
diluted with water and the iodine was removed by washing
with saturated sodium thiosulfate solution (20 mL). The
product was extracted using ethyl acetate (2 ꢁ 20 mL). The
organic layers were combined, washed using saturated sodium
chloride solution (NaCl, 10 mL), dried over sodium sulfate
(Na2SO4), filtrated and evaporated in vacuum. The crude
material was purified by silica gel column chromatography
with petroleum ether/ethyl acetate to yield a pale yellow
powder, S1b (122 mg, 49.09%) (Scheme 1). 1H NMR
(400 MHz, DMSO-d6) ꢀ 10.28 (s, 1H), 7.95 (d, J ¼ 8.8 Hz,
2H), 7.70 (s, 1H), 7.29 (s, 1H), 6.93 (d, J ¼ 8.8 Hz, 2H), 6.78
(s, 1H), 5.30 (t, J ¼ 7.5 Hz, 1H), 3.96 (s, 3H), 3.32 (d,
J ¼ 7.4 Hz, 2H), 1.74 (s, 3H), 1.68 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) d 176.2, 162.4, 161.5, 160.7, 156.0,
132.7, 128.0, 128.0, 123.9, 121.7, 121.4, 116.4, 115.8, 104.6,
99.3, 56.3, 27.6, 25.5, 17.6. LR-ESI-MS m/z: 337.3 [M + H]+;
335.1 [M ꢂ H]+. HR-ESI-MS measured 337.1441 ([M + H]+,
calcd 337.1440 for C21H21O4).
DMSO-d6) 9.07 (s, 1H), 9.02 (s, 1H), 7.47 (s, 1H), 6.90 (s,
1H), 6.78–6.73 (m, 2H), 6.60 (s, 1H), 5.40 (dd, J ¼ 12.7,
3.0 Hz, 1H), 5.23 (t, J ¼ 7.5 Hz, 1H), 3.84 (s, 3H), 3.18 (d,
J ¼ 7.4 Hz, 2H), 3.06 (dd, J ¼ 16.8, 12.7 Hz, 1H), 2.63 (dd,
J ¼ 16.8, 3.0 Hz, 1H), 1.71 (s, 3H), 1.65 (s, 3H). 13C NMR ꢀ
(100 MHz, DMSO-d6) 190.4, 163.4, 161.9, 145.6, 145.2,
132.2, 129.8, 126.0, 123.4, 121.9, 117.9, 115.3, 114.3, 113.5,
99.3, 79.2, 56.1, 43.2, 27.3, 25.6, 17.6. LR-ESI-MS m/z: 355.1
[M + H]+. HR-ESI-MS measured 355.1535 ([M + H]+, calcd
355.1545 for C21H23O5).
Preparation of (E)-4’-hydroxy-7-methoxy-6 -
(4-hydroxy-3-methyl-2-butenyl)-flavanone
The microorganism, Cunninghamella elegans AS 3.2028, was
transferred in turn from slants to conical flasks of 500 mL
containing 150 mL of potato media. The flasks were
incubated at 25 ꢀC with rotary shaking at 160 rpm. After 2
days, 15 mg of a biologically transformable substrate was
dissolved in 1.5 mL acetone, added to each flask, and
incubated with shaking for 5 days. The mixture was filtered,
and extracted for three times with ethyl acetate (3 ꢁ 150 mL).
After a sufficient quantity of the transformed product
accumulated, it was isolated and purified by column chro-
matography or semipreparative HPLC (Scheme 3), to yield a
light yellow amorphous powder, which was confirmed as
C21H22O5 by high-resolution electrospray ionization mass
Synthesis of 30,4’-dihydroxy-7-methoxy-6-(3-methyl-2-
butenyl)-flavanone (S2f)
Chloromethyl methyl ether (714 mg, 8.87 mM) was added
slowly to a stirred solution of S2a (500 mg, 3.62 mM) and
anhydrous K2CO3 (2000 mg, 14.47 mM) in dry acetone
1
spectrometry (HR-ESI-MS) at m/z 377.1371 [M + Na]+. H-
NMR (600 MHz, CD3OD): d 7.60 (s, 1H, H-5), 7.34

DOI: 10.3109/00498254.2015.1074763
Structural elucidation of in vitro metabolites of bavachinin
3
OH
OH
Scheme 1. Reagents and conditions: (a) I2,
DMSO, 90 ꢀC, 3 h (49.09%).
O
O
O
O
O
O
S1a
S1b
OH
OH
O
O
O
O
a
O
OH
O
b
Scheme 2. Reagents and conditions: (a)
MOMCl, K2CO3, acetone, room tempera-
ture, 2 h; (b) (CH3)3SiO-K+, C2H5OH, reflux,
4 h; (c) KF, CH3OH, reflux, 8 h; (d) HCl,
CH3OH, reflux, 10 min.
+
H
H
O
O
S2c
S2a
S2b
O
O
O
O
O
O
O
O
OH
O
O
O
c
O
O
S2d
S2e
OH
OH
O
O
d
O
S2f
OH
Scheme 3. Biotransformation of bavachinin
by C. elegans AS 3.2028.
OH
O
O
O
O
O
O
C. elegans AS 3.2028
HO
(d, J ¼ 8.5 Hz, 2H, H-20,60), 6.82 (d, J ¼ 8.5 Hz, 2H, H-30, 50),
6.55 (s, 1H, H-8), 5.55 (t, 1H, H-200), 5.40 (dd, J ¼ 13.2,
2.9 Hz, 1H, H-2), 3.97 (s, 2H, H-400), 3.88 (s, 3H, H-12), 3.07
(dd, J ¼ 16.9, 13.2 Hz, 1H, H-3 a), 2.71 (dd, J ¼ 16.9, 2.9 Hz,
1H, H-3 b), 1.74 (s, 3H, H-500). 13C-NMR (150 MHz,
CD3OD): d 193.8 (C-4), 165.9 (C-7), 164.5 (C-9), 159.0 (C-
40), 137.4 (C-300), 131.3 (C-10), 129.0 (C-5), 127.8 (C-20, 60),
125.3 (C-200), 123.9 (C-6), 116.3 (C-10), 114.9 (C-30, 50)
100.0 (C-8), 81.3 (C-2), 68.8 (C-400), 56.5 (C-12), 44.9 (C-3),
28.3 (C-100), 13.7 (C-500).
Microsomal incubation procedure
A mixture (0.1 mL) containing 0.5 mg/mL of rat liver
microsomes, 200 mM bavachinin, 10 mM MgCl2 and 2 mM
NADPH in 50 mM Tris-HCl buffer (PH 7.4) was incubated at
37 ꢀC in a temperature-controlled water bath for 60 min. The
mixture was preincubated for 5 min at 37 ꢀC prior to NADPH
addition. The reaction was stopped using 0.1 mL cold
methanol to precipitate the proteins. The reaction vial was
stored at ꢂ20 ꢀC for 30 min. Subsequently, the samples were
centrifuged and the supernatants were analyzed by LC-MS.
Control samples were incubated without substrate or without
NADPH (Nikolic et al., 2004).
Rat liver microsomes
Rats were individually weighed and decollated to prepare
liver microsomes. Livers were perfused using ice-cold saline
followed by mincing and homogenizing with three volumes of
100 mM phosphate buffer (pH 7.4, containing 0.15 M KCl).
The homogenates were centrifuged at 9000 g for 30 min at
4 ꢀC. Subsequently, the supernatants were centrifuged at
105 000g for 60 min at 4 ꢀC to isolate liver microsomes, which
were resuspended in 100 mM phosphate buffer (K2HPO4-
KH2PO4, pH 7.4) and stored at ꢂ80 ꢀC until further use
(Bi et al., 2015).
Apparatus and analytical conditions
Analyses were performed using a Shimadzu UFLC-XR HPLC
system (Shimadzu Scientific Inc., Kyoto, Japan) coupled with
a LCQ ion trap mass spectrometer system (Thermo Fisher
Scientific, Waltham, MA). An XSELECT HSS T3 column
(2.1 mm ꢁ 150 mm, 3.5 mm) (Waters, Milford, MA) was used
for the LC analysis. Metabolite separation was conducted
under the following conditions: flow rate, 0.2 mL/min; solvent
A, 0.05% HCOOH; solvent B, CH3CN; 0–8 min, 40–74% B;

4
F. Xie et al.
Xenobiotica, Early Online: 1–11
Figure 2. Computer-reconstructed, selected ion chromatogram for the positive ion electrospray LC-MS analysis of an incubation of bavachinin and
pooled rat liver microsomes. (A) 60 min (B) 0 min. Selected ions were m/z 337, 339, 355.
8–13 min, 74–80% B; 13–18 min, 80–90% B (v/v). ESI was
performed in the positive mode at a spray voltage of 4.5 kV;
the capillary voltage and tube lens were set at ꢂ45 V and
ꢂ125 V, respectively. The sheath gas flow rate was 40
(arbitrary units) and the aux gas flow rate was 10 (arbitrary
units). The capillary temperature was set to 320 ꢀC.
Normalized collision energy of 20–35% was applied to ions
for the MSn experiments.
0 min (Figure 2B) and increased with time. The MSn
experiments were subsequently employed to analyze the
fragmentation pathways of bavachinin and the eight metab-
olites. Positive ESI MSn spectra are presented in Figure 3.
Mass spectral fragmentation of bavachinin
A molecular ion peak [M + H]+ at m/z 339 was seen in the
positive ESI full-scan mass spectrum of bavachinin. In the
MS2 spectrum, the ion at m/z 321 was produced by the
elimination of an H2O molecule from the ion at m/z 339 (Ma
et al., 1997), and two abundant fragment ions at m/z 283 and
271 were generated (Figure 3A). The ion at m/z 271 was
produced by the elimination of the prenyl chain from the ion
at m/z 339 (Xu et al., 2012), whereas the ion at m/z 283 was
produced by the loss of isobutylene chain [(CH3)2 C ¼ CH2]
from the ion at m/z 339 (Su et al., 2015), which exhibited a
stable benzyl structure (Nikolic et al., 2004). The preceding
two pathways can be used for the inference of metabolism
occurring in the prenyl group. The product ions at m/z 283
and 271 fragmented into ions at m/z 189 and 177 by ring B
elimination (Simons et al., 2009). According to a previous
report (Fabre et al., 2001), bavachinin can also produce a
Results and discussion
Metabolism of bavachinin in rat liver microsomes
The computer-reconstructed, selected ion chromatogram (m/z
337, 339, 355) for the positive ion electrospray LC-MS
analysis of the incubation mixture of bavachinin, and pooled
liver microsomes is shown in Figure 2(A). Compared with the
control groups, eight peaks were eluted before bavachinin (tR
11.7 min) in the LC-MS profile, including five major peaks
(metabolite 1, tR 6.6 min; metabolite 2, tR 6.8 min; metabolite
3, tR 8.2 min; metabolite 4, tR 10.3 min; metabolite 5, tR
10.7 min), three minor peaks (metabolite 6, tR 9.3 min;
metabolite 7, tR 9.6 min; metabolite 8, tR 11.1 min). These
eight metabolites were absent from the incubation mixture at

DOI: 10.3109/00498254.2015.1074763
Structural elucidation of in vitro metabolites of bavachinin
5
Figure 3. The MS2 spectra of protonated bavachinin (A), M1 (B), M2 (C), M3 (D), M4 (E), M5 (F), M6 (G), M7 (H) and M8 (I) obtained by ion trap
mass spectrometry. The MS3 spectra of the ions at m/z 337 of M1 (B-a) and M2 (C-a), m/z 283 of M3 (D-a) obtained through ion trap mass
spectrometry.

6
F. Xie et al.
Xenobiotica, Early Online: 1–11
Figure 3. Continued.

DOI: 10.3109/00498254.2015.1074763
Structural elucidation of in vitro metabolites of bavachinin
7
Figure 3. Continued.

8
F. Xie et al.
Xenobiotica, Early Online: 1–11
Figure 3. Continued.
typical fragment ion (1,3A+ ion) of flavonoids at m/z 219 by a
retro-Diels-Alder (RDA) reaction on ring C. In addition, 1,4B+
ion at m/z 147 was formed by a cleavage of bond 1,4 on ring C
(Simons et al., 2009). The main fragmentation pathways are
illustrated in Figure 4.
corresponded to losses of 71–72 mass units, which might be
containing four carbon atoms and one oxygen atom; the
additional oxygen added to M3 could not be located on the
benzylic carbon (Nikolic et al., 2004). The ion at m/z 283 was
fragmented through the RDA reaction on ring C to form the
ion at m/z 163. In addition, 1,4B+ ion at m/z 147 was also
formed through the cleavage of bond 1,4 at ring C as
bavachinin (Simons et al., 2009). These major fragmentation
pathways indicate that the prenyl chain was oxidized. On the
basis of several relevant studies (Nikolic et al., 2004, 2005),
we speculate that C-200 of the prenyl chain was oxidized, thus
yielding a more stable structure.
Identification of the metabolites of bavachinin
In the positive ESI full-scan mass spectrum, metabolites
M1-4 and M6-8 produced the same protonated molecule
[M + H]+ at m/z 355, whereas metabolite M5 produced an ion
at m/z 337. The mass measurements of M1-4 and M6-8
corresponded to an elemental composition of C21H22O5,
suggesting that they were hydroxylated products of bavachi-
nin. The molecular mass and retention time of M5 indicate
that it may be an oxidative product of bavachinin.
In the MS2 spectrum of M1 (Figure 3B), the ions at m/z
337 and 299 were the most abundant fragment ions of m/z
355. The ion at m/z 299 was formed by the keto group
elimination of ring C and by the loss of the CO group of ring
B (Zhang et al., 2014). The ion at m/z 337 may have formed
by eliminating an H2O molecule from M1 (Ma et al., 1997).
In addition, the ion at m/z 147 was formed, indicating that ring
B was intact (Simons et al., 2009). Moreover, ion at m/z 217
was produced by the RDA reaction on ring C of the ion at m/z
337 (Fabre et al., 2001), suggesting that the prenyl chain was
oxidized. M1 and M2 may be cis-trans isomers because of
similar product ion mass spectra (Figure 3C). M2 was
identified as trans 40-hydroxy-7-methoxy-6-(4-hydroxy-3-
methyl-2-butenyl)-flavanone by comparing the retention
time in the TIC and molecular weight with those of synthetic
trans 40-hydroxy-7-methoxy-6-(4-hydroxy-3-methyl-2-bute-
nyl)-flavanone (Ma et al., 2015), whereas M1 could be the
geometric isomer of M2, cis 40-hydroxy-7-methoxy-6-(4-
hydroxy-3-methyl-2-butenyl)-flavanone.
Several fragmentation pathways of M4 were similar to
those of bavachinin. For example, based on the loss of H2O,
the ion of m/z 355 produced an ion of m/z 337 (Ma et al.,
1997) (Figure 3E). The ions at m/z 299, 287 and 163 were the
three most abundant fragment ions. The ion at m/z 299 was
produced by the loss of isobutylene chain [(CH3)2C ¼ CH2]
from the ion at m/z 355 (Su et al., 2015), whereas the ion at m/
z 287 was formed by the elimination of prenyl chain from the
ion at m/z 355 (Xu et al., 2012). The ion at m/z 163 (1,4B+)
was also formed by a cleavage of bond 1,4 at ring C as
bavachinin (Simons et al., 2009). Finally, ring C underwent a
RDA reaction to form the ion of m/z 219. The fragmentation
pathways indicate that ring B may have been oxidized;. the
synthetic 30,40-dihydroxy-7-methoxy-6-(3-methyl-2-butenyl)-
flavanone further supported this observation. Many fragmen-
tation pathways of M4 and M6 were similar (Figure 3G).
Moreover, M6 generated a main fragment ion at m/z 245
through a cleavage of the single bond between ring B and ring
C, indicating that ring B was oxidized. Thus, M6 was
identified as 20,40-dihydroxy-7-methoxy-6-(3-methyl-2-bute-
nyl)-flavanone.
M5 produced a protonated molecule at m/z 337 during
positive ion electrospray, and was consided to be an oxidative
product of bavachinin. The ions at m/z 281 and 269 in the
MS2 spectrum (Figure 3F) corresponded to m/z 283 and 271
in the MS2 spectrum of bavachinin. The ion at m/z 269 was
formed through the elimination of the prenyl chain from the
ion of m/z 337, whereas the ion at m/z 281 was produced
The mass spectrum of M3 revealed little information
(Figure 3D). The loss of water molecule (m/z 337) from the
ion at m/z 355 suggested the presence of a hydroxyl group
near a double bond for the formation of a stable conjugated
structure. The fragment ion at m/z 283 and the ion at m/z 284

DOI: 10.3109/00498254.2015.1074763
Structural elucidation of in vitro metabolites of bavachinin
9
+
Figure 4. Main fragmention pathways of
bavachinin.
+H
OH
OH
O
O
O
O
+
O
1,4
+
B
1,3
O
+
+
A
m/z 147
m/z 219
m/z 321
RDA
-H O
2
+
+H
OH
O
O
O
m/z 339
- isobutylene
-prenyl
+
+H
OH
OH
O
O
O
O
O
+
O
m/z 271
m/z 283
-C H OH
6
5
-C H OH
6
5
+
+H
O
O
O
O
+
O
O
m/z 189
m/z 177
OH
OH
O
O
O
OH
O
O
O
OH
M4
OH
O
O
O
M6
HO
M2
OH
OH
O
O
O
OH
O
O
O
O
O
O
OH
M3
M1
HO
OH
OH
OH
O
O
O
O
O
O
OH
O
O
O
M7
M5
OH
M8
Figure 5. Proposed metabolic profile of bavachinin in rat liver microsomes.

10 F. Xie et al.
Xenobiotica, Early Online: 1–11
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through a loss of isobutylene chain [(CH3)2C ¼ CH2] from
the ion at m/z 337. In addition, the presence of fragment ion at
m/z 219 from the RDA reaction, indicated that ring A was
intact. To support this result, 40-hydroxy-7-methoxy-6-(3-
methyl-2-butenyl)-flavone was synthesized, and the retention
time in the TIC and its molecular weight were compared with
those of M5.
Abundant fragmentations in the product ion tandem
mass spectra of M7 and M8 were detected at m/z 219
(Figure 3H, I), suggesting that ring A was intact. In addition,
the elution times of M7 and M8 were different from those for
M4 and M6, indicating that ring C was oxidized. 1,3B+ ion at
m/z 137 and 0,2B+ ion at m/z 121 in the MS2 spectrum of M8
suggested that C-3 was oxidized, whereas M7 may be 2,40-
dihydroxy-7-methoxy-6-(3-methyl-2-butenyl)-flavanone.
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Conclusions
Although prenylated flavonoids occur in various plant
species, little is known about metabolism of these natural
products. Therefore, this study on bavachinin metabolism
develops the understanding of prenylated flavonoid metabol-
ism. In this study, bavachinin was fragmented into eight
metabolites in rat liver microsomes. The structures were
proposed through a LC-ESI-MSn analysis and chemical
synthesis (Figure 5). The results indicate that the eight
metabolites were biotransformed mainly through oxidation.
Our investigation provides much information on the in vitro
metabolism of bavachinin and contributes to the understand-
ing of in vivo bavachinin metabolism.
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prenylflavonoids from Psoralea coryfolia by using Cunninghamella
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xanthohumol and isoxanthohumol, prenylated flavonoids from hops
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Acknowledgements
The authors are grateful to Prof. Wang Chang Hong
(Shanghai R&D Center for Standardization of Traditional
Chinese Medicines) for the gift of liver microsomes.
Declaration of interest
The authors report no conflicts of interest.
This study was supported by a grant of Natural Science
Foundation of Shanghai (13ZR1441700).
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