Anal Bioanal Chem (2006) 386: 264–274
DOI 10.1007/s00216-006-0621-0
ORIGINA L PA PER
Liang Kong . Zhiyuan Yu . Yongming Bao . Xingye Su .
Hanfa Zou . Xin Li
Screening and analysis of an antineoplastic compound in Rhizoma
Chuanxiong by means of in vitro metabolism and HPLC-MS
Received: 13 April 2006 / Revised: 5 June 2006 / Accepted: 13 June 2006 / Published online: 26 July 2006
# Springer-Verlag 2006
Abstract A new screening and analysis method that
combines in vitro metabolism with high-performance
liquid chromatography-mass spectrometry (HPLC-MS)
was developed for the screening and analysis of an
antineoplastic compound, coniferyl ferulate, which is
present in the rhizome of Rhizoma Chuanxiong. Infrared
(IR), ultraviolet visible spectroscopy (UV-Vis), nuclear
magnetic resonance (NMR) and element analysis were
used to identify the molecular structure of coniferyl
ferulate. The quantitative analysis of coniferyl ferulate in
different extracts of Rhizoma Chuanxiong was carried out,
and the metabolism of coniferyl ferulate was investigated
by in vitro incubation with rat liver homogenate. The
metabolite of coniferyl ferulate, ferulic acid ethyl ester, was
identified by HPLC-MS, UV-Vis and IR. In addition,
antineoplastic activities of coniferyl ferulate and ferulic
acid ethyl ester were detected by the MTT assay. The
observed inhibition rate of coniferyl ferulate on the activity
of HeLa cells was over 80% at 5.4 ng μl−1. However, its
metabolite, ferulic acid ethyl ester, showed no antineoplastic activity in vitro.
Keywords Coniferyl ferulate . Ferulic acid ethyl ester .
HPLC-MS . Rhizoma chuanxiong .
Traditional Chinese medicine
L. Kong (*) . Z. Yu . X. Su . H. Zou . X. Li
National Chromatographic R&A Center,
Dalian Institute of Chemical Physics,
The Chinese Academy of Sciences,
Dalian 116023, People’s Republic of China
e-mail: liangkong@dicp.ac.cn
Tel.: +86-411-84379640
Fax: +86-411-84379620
Y. Bao
School of Environmental and Biological Science
and Technology, Dalian University of Technology,
Dalian 116024, People’s Republic of China
Introduction
The indisputable identification of the active components of
a herbal product generally constitutes a bottleneck in the
(commercial) development of natural medicines. Rhizoma
Chuanxiong (English name is Szechwan Lovage Rhizome),
one of the most important crude drugs among the
traditional Chinese medicines (TCMs), is the dried rhizome
of Ligusticum Chanxiong HORT., which belongs to the
Umbelliferae family. Rhizoma Chuanxiong has been
widely used to treat angiocardiopathy [1, 2], nephropathy
and menstrual disorders in clinical applications [3, 4]. It has
also been reported that the extract of Rhizoma Chuanxiong
has antimicrobial activity and can be used as a calming
agent and to treat anemia. However, to date, the active
constituents of this extract have not been clearly identified
[4–9]. The main constituents of the Rhizoma Chuanxiong
extract are essential oil, alkaloid, phenols, lactone, ferulic
acid, among others [4, 10–13]. The principal components
of the essential oil are phthalides, such as ligustilide,
butylphthalide, sankyunolide and neocnidilide [14], which
are reported to strongly increase the peripheral blood flow
[15]. The representative alkaloid of Rhizoma Chuanxiong
is tetramethylpyrazine (chuanxiongzine), which has been
clinically used for treating ischemic cerebrovascular
disease in China and appears to work through a vasodilation mechanism and hematoblast depolymerization [4].
Another alkaloid, perlolyrine, has been reported to be used
in an eutherapeutic treatment for coronary heart disease
[16]. The phenols lactone and ferulic acid have been shown
to have a strong inhibitory action against spasms [17].
The metabolism of some of the components present in
the extract of Rhizoma Chuanxiong, such as ferulic acid,
perlolyrine and tetramethylpyrazine, have recently been
studied [18–22]. Tang et al. reported that perlolyrine, an
alkaloid present in Rhizoma Chuanxiong, is metabolized to
three metabolites in rat, and reported the molecular
structure of these metabolites [20]. The proposed pathway
was also described. Sekiya et al. investigated the metabolic
pathway of butylidenephthalide in the hairless mouse [22].
Most of the metabolic studies on Rhizoma Chuanxiong
265
have been performed with the aim of isolating and
identifying the constituents of its extract. The most
common methods used for analyzing and identifying
components of TCMs or their metabolites are thin-layer
chromatography (TLC), gas chromatography (GC) or gas
chromatography coupled with mass spectrometry (GCMS), high-performance liquid chromatography (HPLC) or
high-performance liquid chromatography coupled with
mass spectrometry (HPLC-MS) and capillary electrophoresis (CE) [10, 18–27]. Since some components in TCMs
are unstable at high temperature, HPLC or HPLC-MS
represent the more suitable methods for analyzing and
preparing thermal instable constituents, especially in
comparison with GC and GC-MS.
We have established analytical methods based on HPLC
and HPLC-MS for the metabolic screening of the active
constituents from Rhizoma Chuanxiong. In doing so, we
have identified a thermally unstable and antineoplastic
compound of coniferyl ferulate [3- (4-hydroxy-3-methoxyphenyl) -acrylic acid 3-(4-hydroxy-3-methoxy-phenyl)allyl ester] from the crude extract of Rhizoma Chuanxiong.
A preliminary biotransformation study of coniferyl ferulate
was also performed in an in vitro experiment with rat liver
homogenate.
Experimental
Chemicals and reagents
Crude medicinal material of Rhizoma Chuanxiong, originating from Sichuan Province in China, was purchased
from a local drug store. Reduced nicotinamide adenine
dinucleotide phosphate (NADPH; Sigma, St. Louis, Mo.)
was used for the rat liver homogenate incubation experiment. Ethanol-D6 (D, 99%) was purchased from the
Cambridge Isotope Laboratories (Andover, Mass.).
HPLC-grade acetonitrile was used; other chemicals were
of analytical grade. Distilled water purified with Milli-Q
water (Millipore, USA) was used for the preparation of all
solutions and dilutions.
Apparatus and instruments
The HPLC system (Shimadzu, Kyoto, Japan) consisted
of two LC-10ADvp pumps, a SIL-10Avp auto-sample
injector, a SPD-M10Avp diode array detector (DAD), a
SCL-10Avp system controller and a Class-vp5.0 chromatography workstation. The preparative chromatography consisted of two LC-8A pumps (Shimadzu), a 7725i
injection valve (Rheodyne, Rohnert Park, Calif.) with a
2-ml loop, a SPD-10Avp UV-Vis detector (Shimadzu)
and a WDL-95 chromatography workstation (National
Chromatographic R&A Center, China).
Analysis was performed on a RP-HPLC column packed
with 5 μm Kromasil C18 (200×4.0 mm ID); the mobile phase
consisted of water and acetonitrile. The linear gradient from
25% acetonitrile/water (v/v) to 85% acetonitrile/water (v/v)
in 90 min was adopted. The flow rate was set at 0.76 ml
min−1. The preparative purification was carried out using a
solid-phase extraction (SPE) cartridge packed with 30 g of
40-μm C18 particles (National Chromatographic R&A
Center), a semi-preparative column A (200×14 mm ID)
and a column B (250×8 mm ID) packed with 5 μm Kromasil C18 particles.
Element analysis was performed using an Elemental
Analyzer MOD1106 (Carlo Erba, Italy).
The HPLC-MS system consisted of an HPLC system
with a LCMS-2010 mass spectrometer (Shimadzu). The
MS analysis was performed on an atmospheric pressure
chemical ionization (APCI) interface with positive and
negative ion detection. The instrument acquisition parameters were set as follows: APCI probe temperature, 400 °C;
CDL temperature, 250 °C (+)/200 °C (−); gas flow,
2.5 l/min; block temperature, 200°C; probe voltage, 4.5 kV
(+)/−4.0 kV (−); CDL voltage, −45 V (+)/45 V (−); Q-array
voltage, 15 V (+)/−15 V (−); Q-array RF, 150; detector
gain, 1.8 kV; scan speed, 1000; m/z range, 50–400.
Preparation of Rhizoma Chuanxiong extract
The dried rhizome of Rhizoma Chuanxiong was ground to
a powder in a grinder, following which 10 g of the powder
was weighed accurately and added to each of 75 ml of
water, methanol, 95% ethanol and ethanol and then heated
to boiling for 1 h. The extracts were filtered through a
0.45-μm membrane and stored for further experiments.
The effect of extracting time on the amount of coniferyl
ferulate extracted from Rhizoma Chuanxiong was investigated by removing 1 ml of the extracting solutions at the
following time points: 15, 30, 45, 60, 75, 105, 135,
195 min. The extracts were then filtered through a 0.45-μm
membrane and stored for further analysis.
Preparation of coniferyl ferulate
A 120-g sample of Rhizoma Chuanxiong was crushed in a
grinder and added to 800 ml ethanol and then heated to
boiling for 1 h. The ethanol extracts were filtered through
a 0.45-μm membrane, following which the filtrates were
collected and the solvents removed at 40 °C by vacuum
evaporation. The residues were further extracted using a
disposable SPE cartridge (30 g, 40-μm C18) previously
conditioned with acetonitrile (90 ml) and water (90 ml),
and the adsorbates were eluted successively with 200 ml
of solutions consisting of 30% and 60% acetonitrile/water
(v/v), respectively. The latter fraction was collected and
the purity of coniferyl ferulate was determined to be about
35%. Consequently, a second purification step was
deemed necessary for structure identification. The semipreparative column A was used to purify the latter fraction
with a mobile phase composed of 50% acetonitrile/water
(v/v), a flow rate of 9.2 ml min−1 and a detection
wavelength of 280 nm. The fraction with a retention time
at 8.5 min was collected and the solvent was removed at
266
40 °C by vacuum evaporation. The residue was dissolved
in 2 ml 80% acetonitrile/water (v/v) for refining. The
semi-preparative column B was used to refine the fraction
of coniferyl ferulate with a mobile phase composed of
37% acetonitrile/water (v/v) and a flow rate of 3 ml min−1,
and the fraction with a retention time at 30.7 min was
collected for structure identification and the metabolism
experiments.
Preparation of the standard solution and the sample
for quantitative analysis
The stock solution was prepared by dissolving 19.7 mg
coniferyl ferulate in 50% acetonitrile/water (v/v), and a
series of standard solutions was prepared by diluting the
stock solution (approx. 3.152–15.76 μg ml−1).
A 3-ml aliquot of the extracting solution of Rhizoma
Chuanxiong was passed through the SPE cartridge
(250 mg, 40-μm C18) previously conditioned with acetonitrile (2.5 ml) and water (2.5 ml). The retained fraction
was eluted by 1 ml acetonitrile, and the acetonitrile fraction
was collected for analysis.
Rat liver homogenate incubation
Male Sprague-Dawley (SD) rats weighing about 300±10 g
were obtained from the Experimental Animal Center,
Dalian Medical University, China. The rats had free access
to food and water prior to experiments.
The rats were killed and their livers rapidly removed,
washed, minced, homogenized with ice-cold KrebsHenseleit buffer [28] (0.1 g liver ml−1 buffer) and centrifuged
at 250 g for 3 min at room temperature. The supernatant was
subsequently pooled for incubation. The concentration of
P450 was detected by spectrophotometer [29].
The incubation medium consisted of Krebs-Henseleit
buffer, 2.7 mg coniferyl ferulate or 20 mg crude extract of
Purification of the metabolite
Following the termination of the incubation experiment,
the medium was centrifuged at 9500 g at room temperature.
The supernatant was diluted with the same volume of water
and passed through a 250-mg C18 or 30-g C18 SPE
cartridge (40 μm) for analytical or preparative preparation,
respectively. The SPE cartridge was washed with 0.4 ml or
40 ml (analysis or preparation, respectively) of a 17%
acetonitrile/water (v/v) solution, and the retained compounds were then eluted with 0.6 ml or 60 ml (analysis or
preparation, respectively) acetonitrile. The acetonitrile
eluate was collected and evaporated under reduced pressure distillation at 35 °C, and the remaining residue was
dissolved with a mobile phase for analysis and preparation.
The semi-preparative column A was used to refine the
metabolite of coniferyl ferulate with a mobile phase of 33%
acetonitrile/water (v/v) and a flow rate of 5.9 ml min−1, and
the fraction with a retention time at 21.9 min was collected
and evaporated under reduced pressure distillation at
35 °C. The remaining residue was identified by ultraviolet-visible and infrared spectroscopy and HPLC-MS.
f1
Abs
Fig. 1 Chromatographic fingerprints for the crude extract of
Rhizoma Chuanxiong before (A)
and after (B) metabolism. Experimental conditions: column,
200×4.0 mm ID packed with
Kromasil C18 (5 μm); mobile
phase, a linear gradient from
25% acetonitrile/water (v/v)
to 85% acetonitrile/water (v/v)
in 90 min; flow rate, 0.76 ml
min−1; UV detection wavelength, 316 nm. Peak f1 Coniferyl ferulate, peak m1 ferulic
acid ethyl ester
Rhizoma Chuanxiong, 1 mM NADPH and various mounts of
liver homogenate in a total volume of 3 ml. Coniferyl ferulate
or the crude extract of Rhizoma Chuanxiong was incubated for
the indicated time at 37 °C and the incubation terminated by
the addition of 1.5 ml acetonitrile. The blank experiment was
carried out under the same conditions by replacing the liver
homogenate with Krebs-Henseleit buffer.
The recovery of coniferyl ferulate and ferulic acid ethyl
ester was 82.7 and 89.5%, respectively, from the liver
homogenate.
The volume of the incubation experiments with the liver
homogenate was scaled up in order to obtain sufficient
quantities of metabolites for structure determination.
4.0x10
5
2.0x10
5
(a)
m1
(b)
0.0
0
20
40
Retention time (min)
60
267
Results and discussion
MTT [3-(4, 5 dimethylthiazol-2-yl)-2,
5 -dimethyltetrazoloium bromide] assay
Identification of coniferyl ferulate
The HeLa cells were seeded into 96-well microplates at a
density of 103∼104 cells well−1. The test sample dissolved
in culture medium was added into each well (each group
had four wells) and then cultured for 24 h.
Following the incubation, the MTT solution (5 mg ml−1)
was added to each well. The plate was incubated for a
further 3∼4 h at 37 °C, the supernatant was discarded and
DMSO (0.1–0.2 ml) was added to each well. The test plate
was then agitated by a microplate shaker for 10 min.
The optical density at 570 nm was measured by a
microplate reader.
Int.
The first step in our analysis was to investigate the
metabolism of the crude extract of Rhizoma Chuanxiong.
The chromatograpms for the crude extract before and after
metabolism in vitro are shown in Fig. 1. The area of peak f1
in chromatogram A clearly decreased following biotransformation by the rat liver homogenate, and a new peak, m1,
appeared in chromatogram B, suggesting that the substance
at peak f1 was likely to have been metabolized by rat
cytochrome P450 and that peak m1 may be its metabolite.
In order to more clearly determine the metabolic pathway
for rat cytochrome P450-dependent biotransformation of
f1, we purified the fraction of peak f1.
192.90
4.0e6
a
3.0e6
210.90
2.0e6
233.95
1.0e6
149.00
252.00
177.90
220.00
0.0e6
125
150
Int.
175
200
250
386.95
354.95
275.05
225
275
300
325
350
375
m/z
163.00
b
5000e3
204.00
2500e3
103.00
0e3
131.00
144.00
125
172.00
185.00
150
175
213.00
200
236.00
225
325.00
259.00 277.00
250
275
300
352.95
325
350
396.20
375
m/z
c
M1
O
M2
O
H
O
O
HO
O
OH
O
+
HO
H
OH
O
O
Coniferyl ferulate
MW 193
MW 163
MW 356
Fig. 2 HPLC-MS (APCI) spectra of peak f1 using negative (a) and positive (b) ion detection modes and the decomposition mechanism
(c) of coniferyl ferulate (peak f1) during APCI ionization
268
OMe), 3.90 (3 H, s, -OMe). The shift value of the 13CNMR spectrum in CDCl3 was observed as follows: δ(ppm)
167.3 (C-9), 148.2 (C-3), 147.0 (C-4), 146.8 (C-3′), 146.0
(C-4′), 145.3 (C-7), 134.6 (C-7′), 129.0 (C-1′), 127.1 (C-1),
123.3 (C-6), 121.1 (C-8′), 120.8 (C-6′), 115.4 (C-8), 114.9
(C-5′), 114.6 (C-5), 109.5 (C-2), 108.5 (C-2′), 65.4 (C-9′),
56.0 (2×−OMe). With the combined NMR results and the
element analysis of the compound (C%=66.9, H%=5.7 and
O%=27.4), the molecular formula of f1 can be deduced to
be C20H20O6.
We also characterized f1 by its IR spectrum. The major
absorption peaks in the IR spectrum (KBr, cm−1) are as
follows: 3411 (–OH), 3000, 2963, 2935 (CH3–C), 2890,
2842 (CH3–O), 1697 (=CH–CO–O–CH2–,νC=O), 1631
The purity of the obtained fraction was measured as 98%
by normalization of the retained peaks on reversed phase
(RP)-HPLC. Following the vacuum evaporation of the
solvents in the f1 fraction at 35 °C, the 1H/13C-nuclear
magnetic resonance (NMR) spectra of the residue were
collected on a Varian INOVA NMR spectrometer (Varian,
Palo Alto, Calif.). The 1H-NMR spectrum (CDCl3,
400 MHz) was obtained as follows: δ(ppm) 7.67 (1 H, d,
J=16 Hz, H-7), 7.08 (1 H, dd, J=8, 1.8 Hz, H-6), 7.03 (1 H,
d, J=2 Hz, H-2), 6.94 (1 H, d, J=3.2 Hz, H-2′), 6.91 (2 H,
overlapping signals, H-5′/6′), 6.88 (1 H, d, J=8 Hz, H-5),
6.65 (1 H, d, J=16 Hz, H-7′), 6.35 (1 H, d, J=16 Hz, H-8),
6.25 (1 H, dt, J=15.6, 6.8 Hz, H-8′), 5.86 (brs, OH),
5.7 (brs, OH), 4.85 (2 H, d, J=6.8 Hz, H-9′), 3.91 (3 H, s,
Int.
221.00
10.0e6
7500e3
5000e3
2500e3
106.05 134.95
65.00
0e3
50
75
100
125
174.00
150
175
206.00
200
238.95
225
279.05 303.00 329.00
250
275
300
325
385.90
350
375
m/z
6
1.6x10
MS(APCI)
6
1.2x10
mAU
5
UV-Vis
5
m1
4.0x10
f1
1.6x10
6
1.2x10
6
8.0x10
5
4.0x10
5
8.0x10
0.0
200
400
500
600
700
800
700
800
Wavelength (nm)
UV-Vis
mAU
300
4
8.0x10
4
mAU
6.0x10
0.0
4
4.0x10
4
2.0x10
0
10
20
30
Retention time (min)
40
50
0.0
200
300
400
500
600
Wavelength (nm)
Fig. 3 HPLC-DAD analysis of coniferyl ferulate (f1) and its metabolite (m1) of ferulic acid ethyl ester as well as HPLC-MS (APCI,
negative ion detection mode) spectra of m1
269
O
possible decomposition mechanism of peak f1 shown in
Fig. 2c.
O
Identification of the metabolite of coniferyl ferulate
HO
O
Ferulic acid ethyl ester
MW 222
Fig. 4 Molecular structures of ferulic acid ethyl ester (m1)
The extract of the incubation medium for coniferyl ferulate
was analyzed by HPLC-MS, and the obtained chromatograms with UV-Vis spectra are shown in Fig. 3. It can be
seen that the metabolite (peak m1) of coniferyl ferulate was
less highly retained – or more polar – than coniferyl
ferulate (peak f1) itself, and a molecular ion peak
corresponding to m/z 221 [M-H]− of m1 in the negative
ion scan mode was observed (Fig. 3). Following purification of the metabolite fraction by SPE and semi-preparative
HPLC, the major absorption peaks in the IR spectrum
(KBr, cm−1) were as follows: 3450 (–OH), 2970, 2928
(CH3–C), 2866, 2852 (CH3O–), 1702 (=CH–CO–O–
CH2–,νC=O), 1625 (phenyl -CH=CH–, νC=C), 1603,
1513, 1438 (phenyl, νC=C), 930, 846, 811 (1,2,4trisubstituent benzene, δ=C-H), 1269, 1180, 1155 (–CO–
C–O–, νC-O). Based on the above structural information
from the MS, IR and UV-Vis spectra, and following a
search in the data of the Sadtler Standard Spectra series, the
extract could be identified as ferulic acid ethyl ester [33] .
Its molecular structure is also shown in Fig. 4.
(phenyl –CH=CH–,νC=C), 1593, 1515, 1451 (phenyl, νC=C),
910, 852, 816 (1,2,4-trisubstituent benzene, δ=C-H), 1269,
1158 (–CO–C–O–,νC-O).
In order to further validate the structure of f1, HPLC-MS
(APCI) with negative and positive ion detection was
applied for the structure identification. Because of thermal
instability, the compound of peak f1 decomposed and
generated two fragment ions in MS spectra, as shown in
Fig. 2a and b, under positive and negative scan modes,
respectively. It is clearly indicated that the fragment ion of
f1 under negative ion detection mode is 192.90 [M1]−, the Quantification of coniferyl ferulate
ion peak at m/z 210.90 is characterized as [M1+H2O]−, the in Rhizoma Chuanxiong
m/z 233.95 is characterized as [M1+CH3CN]− and the m/z
252.00 is characterized as [M1+CH3CN+H2O]−. The As a method for the quantitative analysis of coniferyl ferulate
fragment ion of f1 under positive ion detection mode is in Rhizoma Chuanxiong had not yet been reported at the time
163.00 [M2]+, and the m/z 204.00 is characterized as of this analysis, one of our aims was to develop such an
[M2+CH3CN]+. Thereby, we were able to calculate the analytical procedure. Standard solutions of coniferyl ferulate
were diluted in the mobile phase at concentrations ranging
molecule weight of f1 to be 356 (M1+M2).
Using the above results and comparing these with from 3.152 to 15.76 μg ml−1. The calibration curve for
reference substances reported in the literature [10, 30–32], coniferyl ferulate was determined as follows:
we were able to identify compound f1 as coniferyl ferulate.
The chemical structure of coniferyl ferulate is shown in C ¼ 2:346 104 4:504 107 A ð0:385 0:87Þ;
Fig. 2c, and its UV-Vis spectrum (Fig. 3) further confirms
the validity of the identity of the molecular structure. The R ¼ 0:9999; n ¼ 5; P < 0:0001ð316 nmÞ
thermal instability of coniferyl ferulate determined here is
consistent with results previously reported, but its decom- where C (ng ml−1) and A represent the concentrations and
position mechanism was not elucidated in the earlier peak areas of coniferyl ferulate, respectively. The repeatHPLC-MS (APCI) analysis because its positive fragment ability and precision of the procedure were investigated,
ion was not detected [10]. In the present study, two and the results are presented in Table 1. The limit of
fragment ions of coniferyl ferulate were observed simul- detection for coniferyl ferulate was about 20 ng.
taneously in the MS spectra, thereby producing the
Table 1 Precision and recovery
for the quantitative determination of coniferyl ferulate
Injection Measured
amount
(μM)
Average
amount
(μM)
Relative standard
deviation (RSD)
1
2
3
19.03
0.33%
19.01
19.10
18.98
Solid-phase extraction
(SPE)
Actual
amount
(μM)
Average of
recovery (n=3)
RSD
75.59%
2.23% 25.18
270
In order to determine the actual content of coniferyl
ferulate in Rhizoma Chuanxiong, we investigated the effect
of varying the extracting solvents and extracting time on
the extracted amount of coniferyl ferulate. Figure 5 shows
the peak area of coniferyl ferulate as it changes with the
length of the extracting time in four different solvents:
ethanol, 95% ethanol, methanol and water. The extracting
solvents and the length of extracting time had some
influence on the extracted amount of coniferyl ferulate. The
peak area of coniferyl ferulate in the ethanol and 95%
ethanol extracts reached a maximum after 60 min of
extraction, while the maximum concentration of coniferyl
ferulate was reached at 30 min in the methanol extract. The
amount of coniferyl ferulate extracted by water was
relatively low, but slightly increased with increasing
extracting times. From Fig. 5 it is quite clear that ethanol
is the best of the four solvents tested with respect to
maximum efficiency in extracting coniferyl ferulate and
that coniferyl ferulate can be extracted from the Rhizoma
Chuanxiong extract within 60 min. If the extracting time
surpassed 60 min, the peak area of coniferyl ferulate
gradually decreased as a result of thermal decomposition.
The results of our quantitative analysis revealed that
ethanol is the best solvent for extracting coniferyl ferulate
from Rhizoma Chuanxiong and that 60 min is the optimal
extraction time. The content of coniferyl ferulate in
Rhizoma Chuanxiong was determined to be 89.64 ppm
under these optimal extraction conditions.
identified by HPLC-MS, UV-Vis and IR. However, this
metabolite does not belong to the cytochrome P450dependent biotransformation pathway. We propose three
possible mechanisms for the metabolism of coniferyl
ferulate during its incubation with the liver homogenate
(Fig. 6). Lipases are likely to catalyze the transesterification
in the presence of a small quantity of ethanol, which was
introduced from unknown sources. However, the results
were reproducible in our laboratory. To further demonstrate
the transesterification process, we introduced ethanol-D6
into the incubation medium. The results of this latter
incubation confirmed our hypothesis to some extent: an
esterifying reaction does occur between ethanol and the
metabolite of coniferyl ferulate (Fig. 7).
Under the conditions of our experiment, coniferyl
ferulate was transformed rapidly by the liver homogenate
of male SD rats. When the concentration of coniferyl
ferulate was plotted against continuous administration
metabolizing time (Fig. 8), the peak area of coniferyl
ferulate decreased by 96% after an incubation of 70 min.
The quantity of a substrate is a critical factor in drug
metabolism in vitro. We therefore investigated whether the
metabolite profile of coniferyl ferulate was influenced by
an increase in the concentration of the rat liver homogenate
during the incubation. A quantitative change was observed
in that the peak area of the metabolite gradually increased
with the concentration of the rat liver homogenate (Fig. 9),
with the concentration of the metabolite reaching a
maximum when the concentration of liver homogenate
was about 20 mg ml−1.
Metabolism of coniferyl ferulate in vitro
by rat liver homogenate
Incubation of the Rhizoma Chuanxiong extract or coniferyl
ferulate with freshly prepared rat liver homogenate resulted
in the biotransformation of coniferyl ferulate and the
formation of a metabolite, which was subsequently
Fig. 5 Effect of the extracting
time plotted against the extracted amounts of coniferyl
ferulate from Rhizoma
Chuanxiong using
different solvents
Antineoplastic activity of coniferyl ferulate
and ferulic acid ethyl ester
Because the bioactivity of coniferyl ferulate has not
been clearly elucidated, we explored its pharmacologi-
271
O
O
Fig. 6 Three possible mechanisms for the metabolism of
coniferyl ferulate in vitro
O
O
HO
O
O
HO
OH
OH
O
O
Lipase
Lipase
CH3CH2OH
O
O
O
+
O
OH
HO
HO
OH
+
O
HO
HO
OH
O
O
P450
CH3CH2OH
P450
CH3CH2OH
O
O
O
O
HO
HO
O
HO
OH
CH3CH2OH
O
OH
O
b
a
O
O
O
HO
OH
O
P450
O
OH
O
O
HO
OH
O
O
O
OH
+
O
H
OH
HO
O
P450
CH3CH2OH
O
O
HO
O
c
cal activity using the test of cellular poison by
measuring its inhibitory activity on HeLa cells with
the MTT assay. Coniferyl ferulate had a considerable
inhibitory effect on the activity of HeLa cells, as
shown in Fig. 10. The inhibition rate of HeLa cell
activity was above 80% following the treatment of the
cells with coniferyl ferulate at a concentration of more
than 5.4 ng μl−1 for 24 h. However, its metabolite,
ferulic acid ethyl ester, did not show any antineoplastic
activity under the same conditions. The antineoplastic
activity of the crude Rhizoma Chuanxiong extract and
its metabolites were also tested by MTT assay. The
antineoplastic activity of the crude extract disappeared
after metabolism in vitro, which is consistent with that
of coniferyl ferulate and its metabolite. The crude
Rhizoma Chuanxiong extract had obvious antineoplastic activity, whereas its metabolites had no antineoplastic activity. Based on the above results, we can
conclude that coniferyl ferulate may have acute tumorinhibition activity in vivo because it is rapidly
272
Inten.
(x100,000)
5.0
O
226.10
4.0
D2
C
O
CD3
3.0
HO
2.0
O
1.0
0.0
100.0
MW 227
205.05
125.0
150.0
175.0
200.0
225.0
250.0
275.0
300.0
325.0
350.0
375.0
400.0
m/z
Fig. 7 HPLC-MS (APCI, negative ion detection mode) spectra of the deuterated ferulic acid ethyl ester
biotransformed into ferulic acid ethyl ester by P450 in
the liver. As a result of its acute antineoplastic activity
in vivo, Rhizoma Chuanxiong has seldom been used
for treating tumors clinically.
Conclusion
An antineoplastic compound, coniferyl ferulate, was
extracted from Rhizoma Chuanxiong and subsequently
screened, purified and identified by HPLC-MS, UV, IR,
NMR and element analysis. At the same time, an HPLC
method has been developed for the quantitative analysis of
coniferyl ferulate in Rhizoma Chuanxiong. The biotransformation of coniferyl ferulate in vitro was studied by
incubation with SD rat liver homogenate, and its metabolite, ferulic acid ethyl ester, was also identified with the
combined usage of HPLC-MS, UV and IR. Coniferyl
ferulate had a considerable acute inhibitory effect on the
activity of HeLa cells, as determined with the MTT assay,
but ferulic acid ethyl ester did not show any antineoplastic
activity.
5
Fig. 8 Plot of peak area of
coniferyl ferulate against
continuous administration
metabolizing time
3.5x10
5
3.0x10
5
2.5x10
Peak area
5
2.0x10
5
1.5x10
5
1.0x10
4
5.0x10
0.0
0
10
20
30
40
Time (min)
50
60
70
80
273
6
Fig. 9 Plot of peak area of
ferulic acid ethyl ester against
the concentration of rat liver
homogenate
6x10
6
5x10
6
Peak area
4x10
6
3x10
6
2x10
6
1x10
0
0
5
10
15
20
25
30
35
40
Concentration (mg/mL)
Fig. 10 Inhibition of coniferyl
ferulate and ferulic acid ethyl
ester on HeLa cell activity
coniferyl ferulate
ferulic acid ethyl ester
100
Inhibition rate (%)
80
60
40
20
0
0
2
4
6
8
10
12
Concentration (ng/µL)
Acknowledgements The financial supports from the Natural
Science Foundation of China (No.90209056) to L.K. and the
Knowledge Innovation program of CAS (KGCX2-SW-213-04) to
H.Z. are gratefully thanked. We also thank Prof. Zongbao Zhao for
his suggestions.
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