Toxicology 289 (2011) 11–18

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Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Evidence for aconitine-induced inhibition of delayed rectifier K+ current in Jurkat

T-lymphocytes
Sheng-Nan Wu a,b,c,∗ , Bing-Shuo Chen c , Yi-Ching Lo d
a
Department of Physiology, National Cheng Kung University Medical College, Tainan City, Taiwan
b
Department of Anatomy and Cell Biology, National Cheng Kung University Medical College, Tainan City, Taiwan
c
Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan City, Taiwan
d
Department of Pharmacology, Kaohsiung Medical University, Kaohsiung City, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Aconitine (ACO) is a highly toxic diterpenoid alkaloid and known to exert the immunomodulatory action.
Received 13 June 2011 However, whether it has any effects on ion currents in immune cells remains unknown. The effects of ACO
Received in revised form 4 July 2011 and other related compounds on ion currents in Jurkat T-lymphocytes were investigated in this study. ACO
Accepted 6 July 2011
suppressed the amplitude of delayed-rectifier K+ current (IK(DR) ) in a time- and concentration-dependent
Available online 18 July 2011
manner. Margatoxin (100 nM), a specific blocker of KV 1.3-encoded current, decreased the IK(DR) amplitude
in these cells and the ACO-induced inhibition of IK(DR) was not reversed by 1-ethyl-2-benzimidazolinone
Keywords:
(30 ␮M) or nicotine (10 ␮M). The IC50 value for ACO-mediated inhibition of IK(DR) was 5.6 ␮M. ACO accel-
Aconitine
Lymphocyte
erated the inactivation of IK(DR) with no change in the activation rate of this current. Increasing the ACO
K+ current concentration not only reduced the IK(DR) amplitude, but also accelerated the inactivation time course of
Inactivation kinetics the current. With the aid of minimal binding scheme, the inhibitory action of ACO on IK(DR) was estimated
with a dissociation constant of 6.8 ␮M. ACO also shifted the inactivation curve of IK(DR) to a hyperpo-
larized potential with no change in the slope factor. Cumulative inactivation for IK(DR) was enhanced in
the presence of ACO. In Jurkat cells incubated with amiloride (30 ␮M), the ACO-induced inhibition of
IK(DR) remained unaltered. In RAW 264.7 murine macrophages, ACO did not modify the kinetics of IK(DR) ,
although it suppressed IK(DR) amplitude. Taken together, these effects can significantly contribute to its
action on functional activity of immune cells if similar results are found in vivo.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ␣7 -nicotinic receptors (Hardick et al., 1995; Mogg et al., 2002;

Aconitine (ACO) is an intensely poisonous alkaloid derived from
plant species Aconitium (Ranunculaceae) (Singhuber et al., 2009;
Borcsa et al., 2011; Xu et al., 2011). ACO-containing herbal extracts
have been demonstrated to exert the inhibition of proliferation in
a variety of neoplastic cells (Chodoeva et al., 2005; Garmanchouk
et al., 2005; Dasyukevich and Solyanik, 2007; Yan et al., 2007)
and have serious toxicity in rat embryos (Xiao et al., 2007) and
in protozoans (Tang et al., 2010). ACO and methyllycaconitine,
its structurally related analog, were reported to be antagonists of
Abbreviations: ACO, aconitine; 1-EBIO, 1-ethyl-2-benzimidazolinone; FBS, fetal
bovine serum; IC50 , the concentration required for a 50% inhibition; IKCa chan-
nel, intermediate-conductance Ca2+ -activated K+ channel; IK(DR) , delayed-rectifier
K+ current; I–V, current–voltage; KD , dissociation constant; KV channel, voltage-
dependent K+ channel; MgTx, margatoxin; SEM, standard error of mean; SSR, sum
of squared residuals.
∗ Corresponding author at: Department of Physiology, National Cheng Kung Uni-
versity Medical College, No. 1, University Road, Tainan City 70101, Taiwan.
Tel.: +886 6 2353535x5334; fax: +886 6 2362780.
E-mail address: snwu@mail.ncku.edu.tw (S.-N. Wu).
Ivy Carroll et al., 2007; De Rosa et al., 2009). Noroxoaconitine,
another structurally related compound, was recently described to
be an inhibitor of mitogen-activated protein kinase (Kostenko et al.,
2011). In our laboratory, we reported that this compound can block
ultrarapid-delayed rectifier K+ currents in heart-derived H9C2 car-
diac myoblasts. However, little information has been reported
concerning the effects of ACO on ion currents in immune cells,
although it could have immunomodulatory actions (Makino et al.,
2009; Singhuber et al., 2009; Zhang et al., 2011).
Voltage-dependent K+ (KV ) channels are recognized to play a
crucial role in the repolarization of membrane potential in nerve
and muscle, controlling their excitability. Due to the maintenance
of membrane potential, they are involved in the proliferation and
activation of immune cells (Chandy et al., 2004; Panyi, 2005;
Gilhar et al., 2011). Specifically, KV 1.3 performs a key function in
the immune system, controlling and leading to proliferation and
interleukin-2 synthesis (Chung and Schlichter, 1997; Beeton et al.,
2005; Gilhar et al., 2011). Effects memory T-cells, which express
high levels of KV 1.3 (Helms et al., 1997; Villalonga et al., 2010),
are present in autoimmune disorders. KV 1.3 channel was reported
to show more specificity for autoreactive T cells than any molec-

0300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2011.07.003
12 S.-N. Wu et al. / Toxicology 289 (2011) 11–18
ular target expressed on all T cells (Beeton et al., 2005; Gilhar
et al., 2011). Besides that, several important studies have demon-
strated that antagonizing the activity of KV 1.3 with either highly
specific peptides or small-molecule channel blockers can reverse
and prevent experimental autoimmune encephalomyelitis in rats
(Vennekamp et al., 2004; Beeton et al., 2005; Pegoraro et al., 2009;
Gilhar et al., 2011).
The Jurkat T cell line, a CD45-deficient clone derived from the
E6-1 clone of Jurkat human T cell leukemic cell line, was demon-
strated to express KV 1.3-type IK(DR) (Pang et al., 2010; Villalonga
et al., 2010). These cells can produce large amounts of interleukin-
2 after treatment with phorbol esters. The KV 1.3 K+ channel, that
exhibits unique gating properties and voltage dependency, has
been described to be an important contributor to the IK(DR) in
immune cells (Wu et al., 1998; Pang et al., 2010; Panyi, 2005;
Villalonga et al., 2010; Gilhar et al., 2011). Therefore, in this study,
we used the Jurkat T leukemia cell line as a model and clearly
demonstrated that ACO could suppress IK(DR) in these cells in a
concentration- and time-dependent fashion. Our results provide
important insights on the still mysterious biological effects of this
compound (Singhuber et al., 2009).
2. Materials and methods
2.1. Drugs and solutions
ACO (aconitine or acetylbenzoylaconine, C34 H47 NO11 ), nicotine and
tetrodotoxin were purchased from Sigma Chemicals (St. Louis, MO, USA).
The purity of ACO was over 99%. ACO was dissolved in dimethyl sulfoxide and made
immediately before the experiments. Amiloride, 1-ethyl-2-benzimidazolinone
(1-EBIO), methyllycaconitine were obtained from Tocris Cookson, Ltd. (Bristol, UK),
and margatoxin (MgTx) was purchased from Alomone Labs (Jerusalem, Israel).
All culture media, FBS, l-glutamine, trypsin/EDTA, penicillin–streptomycin were
obtained from Invitrogen (Carlsbad, CA, USA). Reagent water that was obtained
using a Milli-Q Ultrapure Water Purification System (Millipore, Bedford, MA,
USA) was used in all experiments. The composition of bath solution (i.e., normal
Tyrode’s solution) was as follows (in mM): NaCl 136.5, KCl 5.4, CaCl2 1.8, MgCl2
0.53, glucose 5.5, and HEPES-NaOH buffer 5.5 (pH 7.4). To record K+ currents and
avoid contamination of Cl− current (Maldonado et al., 1991), the patch pipette was
filled with a solution (mM): K-aspartate 130, KCl 20, KH2 PO4 1, MgCl2 1, Na2 ATP 3,
Na2 GTP 0.1, EGTA 0.1, and HEPES-KOH buffer 5 (pH 7.2).
2.2. Cell preparations
The Jurkat T cell line, a human T cell lymphoblast-like cell line (clone E6-1),
was obtained from the Bioresource Collection and Research Center (BCRC-60255;
Hsinchu, Taiwan), and the murine macrophage cell line RAW 264.7 was obtained
from the American Type Culture Collection (TIB-71; Manassas, VA, USA). Jurkat T
cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated
FBS (v/v), 100 U/ml penicillin and 10 ␮g/ml streptomycin. RAW 264.7 cells were
maintained in Dulbecco’s modified Eagle medium supplemented with 10% heat-
inactivated FBS, 100 U/ml penicillin and 10 ␮g/ml streptomycin (Wu et al., 2011).
They were maintained at 37 ◦ C in a 95% air and 5% CO2 humidified atmosphere. The
viability of these cells was often assessed by the trypan blue dye-exclusion test.
The experiments were made five or six days after cells had been cultured (60–80%
confluence).
2.3. Electrophysiological measurements
Before the experiments, either Jurkat T or RAW 264.7 cells were dissociated
with 1% trypsin/EDTA solution and an aliquot of cell suspension was thereafter
transferred to a recording chamber affixed to the stage of a DM-IL inverted micro-
scope (Leica, Wetzlar, Germany). They were bathed at room temperature (20–25 ◦ C)
in normal Tyrode’s solution containing 1.8 mM CaCl2 . Patch electrodes made from
Kimax-51 capillaries (Kimble Glass, Vineland, NJ, USA) using a PP-830 puller (Nar-
ishige, Tokyo, Japan) had a resistance of 3–5 M when they were filled with different
intracellular solutions described above. Patch clamp recordings were performed in
the whole-cell configuration using an RK-400 amplifier (Bio-Logic, Claix, France)
(Wu et al., 1998; Huang et al., 2011).
2.4. Data analyses
The data were stored online in a TravelMate-6253 computer (Acer, Taipei,
Taiwan) at 10 kHz through a Digidata 1322A interface (Molecular Devices, Sun-
nyvale, CA, USA). The latter device was equipped with an Adaptec SlimSCSI card
(Milpitas, CA, USA) via PCMCIA slot and then controlled by pCLAMP9.2 during elec-
trophysiological recordings (Molecular Devices). Currents were low pass-filtered at
3 kHz. The pCLAMP-generated voltage-step protocols were employed to determine
I–V relationships for ion currents (e.g., IK(DR) ). Ion currents were analyzed by using
Origin 8.0 (OriginLab, Northampton, MA, USA) or custom-made macros built in an
Excel 2007 spreadsheet running on Windows 7 (Microsoft, Redmond, WA, USA).
The concentration–response data for inhibition of IK(DR) in Jurkat T-lymphocytes
were fitted to the Hill equation. That is,
n
Emax × [C] H
percentage inhibition = n ,
[C] H + ICn50H
where [C] is the ACO concentration, IC50 and nH are the concentration required
for a 50% inhibition and the Hill coefficient, respectively, and Emax is the maximal
inhibition of IK(DR) caused by ACO.
The inhibitory effect of ACO on IK(DR) in Jurkat T-lymphocytes is explained by a
state-dependent blocker that can bind to the open state of the channel according to
a minimal kinetic scheme:
˛ K+1
CO  O · B,
ˇ K−1
where ˛ and ˇ are the voltage-dependent rate constants for the opening and closing
of KV channels, k+1 and k−1 are those for blocking and unblocking by ACO, and [B]
is the ACO concentration. C, O and O·B denote the closed, open, and open-blocked
states, respectively.
The blocking and unblocking rate constants, k+1 and k−1 , were determined from
the inactivation time constants of IK(DR) in response to the depolarizing pulses. Block-
ing and unblocking rate constants were estimated using the relation:
1
= k+1 × [B] + k−1
b
Specifically, k+1 and k−1 , respectively, result from the slope and the y-axis inter-
cept at [B] = 0 of the linear regression interpolating the reciprocal time constant
(1/ b ) versus different ACO concentrations (0.1–10 ␮M). Based on k+1 and k−1 , the
KD value (i.e., k−1 /k+1 ) was generated.
The steady-state inactivation curve of IK(DR) in the absence and presence of ACO
was plotted against the test potential and fit to the Boltzmann equation:
Imax
I= ,
1 + exp((V − V1/2 )/k)
where V is the conditioning potential in mV, V1/2 is the membrane potential for half-
maximal inactivation, and k is the slope factor of inactivation curve for IK(DR) . The
solver subroutine build in Microsoft Excel (Redmond, WA, USA) was used to fit the
data by a least-squares minimization procedure (Kemmer and Keller, 2010).
All values are expressed as the mean ± SEM with sample sizes (n) indicating the
number of cells from which the data were taken, and error bars are plotted as SEM.
The paired or unpaired Student’s t-test and one-way analysis of variance with the
least-significant-difference method for multiple comparisons were used to evaluate
statistical difference among means. To evaluate the values of sum of squared resid-
uals (SSR), confidence interval was estimated with the use of Fisher’s F distribution
(Kemmer and Keller, 2010). A value of p < 0.05 was considered significant. The sta-
tistical analyses in this study were performed in SAS 8.2 software (SAS Institute Inc.,
Cary, NC, USA).
3. Results
3.1. Effect of ACO on IK(DR) in Jurkat T-lymphocytes
The whole-cell configuration of the patch-clamp technique was
initially used to evaluate the effect of ACO on ion currents in
Jurkat T-lymphocytes. To record K+ outward currents and elim-
inate Ca2+ -activated K+ currents, cells were bathed in Ca2+ -free
Tyrode’s solution. When the cell was held at −50 mV and depo-
larizing voltage pulses from −50 to +60 mV in 10-mV increments
were applied with a duration of 300 ms, a family of outward cur-
rents was elicited (Fig. 1). The threshold for elicitation of these
outward currents was around −30 mV, and the magnitude became
larger with greater depolarizations. This population of outward cur-
rents were identified as IK(DR) and to resemble the KV 1.3-encoded
currents (Matteson and Deutsch, 1984; Wu et al., 1998; Panyi,
2005; Villalonga et al., 2010). Notably, 2 min after exposure to ACO
(30 ␮M), the IK(DR) amplitude was greatly reduced at the poten-
tials ranging from −20 to +60 mV. For example, when depolarizing
pulses from −50 to +50 mV were applied, ACO (30 ␮M) significantly
 S.-N. Wu et al. / Toxicology 289 (2011) 11–18 13

A B
50

mV
0 3000 3000

Current amplitude (pA)

-50
0 100 200 300 control ACO
2000 2000
2000
control

1000 1000

1000
Current amplitude (pA)

0 0
-40 0 40 -40 0 40
0 C Memberane potential (mV)
0 100 200 300

Percentage inhibition (%)

100

80
2000
120

SSR (%2)
60
80
ACO 40
1000 40

20 0
4 5 6 7
0 IC50, μM
0
0.1 1 10 100
0 100 200 300
Time (msec) [ACO], μM

Fig. 1. Inhibitory effect of ACO on IK(DR) in Jurkat T-lymphocytes. Cells were bathed in Ca2+ -free Tyrode’s solution and the recording pipette was filled with K+ -containing
solution. (A) Superimposed current traces obtained in the absence (upper) and presence (lower) of 10 ␮M ACO. The cell was depolarized from −50 mV to various potentials
ranging from −50 to +60 mV in 10-mV increments. Current traces shown on the upper part are controls and those on the lower part were obtained 2 min after addition
of ACO (10 ␮M). The uppermost part indicates the voltage protocol examined. (B) Average I–V relations for initial (circle symbols) and late (square symbols) components
of IK(DR) in the absence (left; filled circles) and presence (right; open circles) of 10 ␮M ACO. (C) Concentration–response curve for ACO-induced inhibition of IK(DR) in Jurkat
T-lymphocytes. The amplitude of IK(DR) measured at the end of depolarizing pulses during exposure to ACO was compared with the control value (mean ± SEM, n = 6–12
for each point). The smooth line represents the best fit to a Hill function. The values for IC50 , maximally inhibited percentage of IK(DR) , and the Hill coefficient were 5.6 ␮M,
100%, and 1.1, respectively. The inset in (C) shows confidence assessment of best-fit parameter values (i.e., IC50 ). The parameter range corresponds to the approximate 95%
confidence intervals. Red line marks parameter value at which SSR amounts to 80.0%2 . (For interpretation of the references to color in this figure caption, the reader is referred
to the web version of the article.)

decreased the amplitude of IK(DR) measured at the end of volt- (100 nM), known to be a specific blocker of KV1.3 -encoded cur-
age pulses from 1507 ± 142 to 173 ± 82 pA (n = 9). After washout of rent (Garcia-Calvo et al., 1993; Helms et al., 1997; Lin et al., 2007),
ACO, the current amplitude at +50 mV was returned to 994 ± 102 pA was effective in suppressing IK(DR) amplitude, although it did not
(n = 7). Under current-clamp conditions, ACO (10 ␮M) significantly alter the inactivation time course of this current. Furthermore, nei-
depolarized the resting potential to −38 ± 5 mV from a control of ther 1-EBIO (30 ␮M) nor nicotine (10 ␮M) caused any significant
−45 ± 6 mV (n = 6). effect on ACO-induced inhibition of IK(DR) observed in Jurkat T-
The relationship between the concentration of ACO and the lymphocytes. 1-EBIO is an activator of IKCa channels (Shen et al.,
percentage inhibition of IK(DR) was determined and then con- 2007), while nicotine can bind to acetylcholine receptors in T lym-
structed. In these experiments, each cell was depolarized from phocytes (Razani-Boroujerdi et al., 2007). The results indicated that
−50 to +50 mV with a duration of 300 ms and current amplitudes ACO could inhibit IK(DR) which is sensitive to be blocked by MgTx
were measured at the end of the depolarizing pulses. As illus- and its inhibition was unrelated to the activity of IKCa channels or
trated in Fig. 1C, ACO (0.3–100 ␮M) suppressed IK(DR) amplitude to the binding to acetylcholine receptors.
in a concentration-dependent manner. With the use of a nonlin-
ear least-squares fit to the data, the IC50 value required from the 3.3. Kinetic studies of ACO-induced increase on the rate of IK(DR)
inhibitory effect of ACO on IK(DR) in Jurkat T-lymphocytes was cal- inactivation in Jurkat T-lymphocytes
culated to be 5.6 ␮M, and this agent at a concentration of 100 ␮M
nearly abolished current amplitude. In the SSR plot shown in inset During to cell exposure to ACO, in addition to the decreased
of Fig. 1C, a horizontal line at SSR = 80.0%2 was also made to deter- amplitude of IK(DR) , the inactivation of IK(DR) tended to be notably
mine the two IC50 values. For a 95% confidence interval, the lower increased. To provide more evidence for ACO-induced blocking of
and upper values amounted to 4.60 and 6.85 ␮M, respectively. IK(DR) , the time constants for IK(DR) inactivation in these cells were
Because there was a steep slope on both sides of the minimum, further analyzed. The time courses of current inactivation in the
the IC50 value was determined with a high confidence. Therefore, absence and presence of different ACO concentrations were fitted
our results reflect that ACO can exert a significant action on the by a single exponential function. The concentration dependence
inhibition of IK(DR) in these cells. of IK(DR) decay by ACO is illustrated in Fig. 3. The results showed
that the effects of this agent were noted to be concentration-
3.2. Comparison between effect of MgTx or ACO and those of ACO dependent increase in the rate of current inactivation accompanied
plus 1-EBIO and ACO plus nicotine by a decrease in the residual, steady-state current. For example,
when cells were depolarized from −50 to +50 mV with a duration of
The effects of MgTx, ACO, ACO plus 1-EBIO, and ACO plus 1 s, the inactivation time constants of IK(DR) obtained during expo-
nicotine on the amplitude of IK(DR) in Jurkat T-lymphocytes were sure to 0.3 and 3 ␮M ACO were fitted by a single exponential with
examined and compared. As shown in Fig. 2, addition of MgTx the values of 350 ± 7 ms (n = 7) and 252 ± 6 ms (n = 6), respectively.
14 S.-N. Wu et al. / Toxicology 289 (2011) 11–18

A B
1200

Current amplitude (pA)

1000 a
50

mV

Current amplitude (pA)

b 0 400
800 c -50
0 1000
600 msec
*
* * *
400 200

200

0 0

e
ro

ar

in
AC

BI
nt

ot
M

-E
co
0 500 1000 1500

ic
+1

+n
O

O
Time (msec)

AC

AC
Fig. 2. Comparison in the effects of MgTx, ACO, ACO plus 1-EBIO, and ACO plus nicotine on IK(DR) recorded from Jurkat T-lymphocytes. Cells were bathed in Ca2+ -free Tyrode’s
solution. Each cell was depolarized from −50 to +50 mV with a duration of 1 s, and current amplitudes were measured at the end of each depolarizing pulse. (A) Superimposed
current traces obtained in the absence (a) and presence of 1 nM (b) and 100 nM (c) MgTx. Inset indicated the voltage protocol used. (B) Summary of the data showing effects
of MgTx (100 nM), ACO (10 ␮M), ACO (10 ␮M) plus 1-EBIO (30 ␮M), and ACO (10 ␮M) plus nicotine (10 ␮M) on IK(DR) (mean ± SEM; n = 5–7 for each point). * Significantly
different from control.

Based on the first-order blocking scheme described in Section 2, 3.4. Steady-state inactivation of IK(DR) during the exposure to ACO
the relationship between 1/tb and [B] was noted to be linear with a
correlation coefficient of 0.97 (Fig. 3C). The blocking and unblocking To characterize the inhibitory effect of ACO on IK(DR) , we next
rate constant measured from six to ten different cells were cal- investigated the voltage dependence of the effect of ACO on IK(DR)
culated to be 0.42 s−1 ␮M−1 and 2.82 s−1 , respectively. Thereafter, in Jurkat T-cells. Fig. 4 shows the steady-state inactivation curve
based on these rate constants, a value of 6.8 ␮M for the dissociation of IK(DR) in the presence of ACO (3 ␮M). In these experiments
constant (Kd = k−1 /k+1 ) could be generated. However, the rate con- conducted with double-pulse protocol, a 1-s conditioning pulse
stant of the inverse reaction (i.e., unblocking rate constant [k−1 ]) to different potentials preceded the test pulse to 0 mV from a
showed little dependence on the ACO concentration. The value of holding potential of −50 mV. The relationship between the con-
k−1 was 2.79 s−1 at 3 ␮M and 0.282 s−1 at 10 ␮M. ditioning potentials and the normalized amplitudes of IK(DR) were

A C
2000
Current amplitude (pA)

a 100
1500 c
b
mV

1000 8
-100
0 1000
msec
500

0
6
1/ τb(sec-1)

0 400 800 1200 1600

Time (msec)
B
Current amplitude (pA)

4
1500
a
b
c
1000
2
0 4 8 12
[Aconitine], μM
500

0 200 400 600 800

Time (msec)

Fig. 3. Evaluation of the kinetics of ACO-induced block of IK(DR) in Jurkat T-lymphocytes. In (A), the time courses of current inactivation in the absence (a; black) and presence
of 0.3 ␮M (b; red) and 1 ␮M (c; blue) ACO were well fitted by a single exponential shown in (B). Each cell was depolarized from −50 to +50 mV with a duration of 1 s. The
inset in (A) indicates the voltage protocol used. In (C), the kinetics of ACO-induced block of IK(DR) in Jurkat T cells was evaluated. The reciprocal of inactivation time constant
of IK(DR) (1/ b ) obtained by exponential fits of the trajectory of IK(DR) inactivation was plotted against the ACO concentration. Data points were fitted by a linear regression
shown in blue line, indicating that ACO-induced blocking occurs with a molecularity of 1. Blocking (k+1 ) and unblocking (k−1 ) rate constants, given by the slope and the y-axis
intercept of the interpolated line, were 0.42 s−1 ␮M−1 and 2.82 s−1 , respectively. (For interpretation of the references to color in this figure caption, the reader is referred to
the web version of the article.)
 S.-N. Wu et al. / Toxicology 289 (2011) 11–18 15

A B
0

mV
-50
0 500 1000 1500 2000

1200 control
1.0
800
0.8
Current amplitude (pA)

400
0.6

I / Imax
0
0.4
0 500 1000 1500 2000

0.2
1200
ACO
0.0
800
-60 -40 -20 0 20 40
400 Conditioning potential (mV)

0
0 500 1000 1500 2000
Time (msec)

Fig. 4. Effect of ACO on steady-state inactivation of IK(DR) in Jurkat T-lymphocytes. (A) Superimposed current traces obtained in the absence (upper) and presence (lower)
of 10 ␮M ACO. The conditioning voltage pulses with a duration of 1 s to various membrane potentials ranging from −50 to +30 mV in 10-mV increments were applied from
a holding potential of −50 mV. Following each conditioning pulse, a test pulse to 0 mV with a duration of 1 s was applied to elicit IK(DR) . The uppermost part indicates the
voltage protocol used. (B) Steady-state inactivation of IK(DR) in the absence (䊉) and presence () of 10 ␮M ACO (mean ± SEM; n = 6–9 for each point).

constructed and fitted with a Boltzmann function, as described
in Section 2. In the control, voltage for half-maximal inactiva-
tion (V1/2 ) and corresponding slope factor (k) were −14.9 ± 1.1 mV
and 5.5 ± 0.1 mV (n = 8), respectively, while in the presence of
ACO (10 ␮M), the values of V1/2 and k were −22.7 ± 1.2 mV and
5.1 ± 0.1 mV (n = 8), respectively. Notably, addition of ACO (10 ␮M)
significantly shifted the midpoint of the inactivation curve toward
hyperpolarizing voltage by approximately 8 mV; however, no sig-
nificant change in the slope factor (i.e., k) was detected in the
presence of ACO. Therefore, there was voltage-dependence of
the steady-inactivation curve of IK(DR) during cell exposure to
ACO.
3.6. Effect of ACO on IK(DR) in amiloride-treated Jurkat
T-lymphocytes
ACO is recognized to affect the amplitude of voltage-dependent
Na+ current (Lin et al., 2008; Wu et al., 2008). Earlier reports showed
that Na+ channels, which are sensitive to block by amiloride, are
functionally expressed in lymphocytes (Lai et al., 2000). For these
reasons, we also evaluated whether blockade of Na+ currents can
influence ACO-induced block of IK(DR) in Jurkat T-lymphocytes. In
these experiments, Jurkat T-cells were preincubated with amiloride
(30 ␮M) for six hours. Notably, there was no significant difference in

1600
3.5. ACO-induced increase in cumulative inhibition of IK(DR)
inactivation
Current amplitude (pA)

1200 a
IK(DR) inactivation was reported to accumulate during repeti-
tive short pulses (Marom et al., 1993). Therefore, in another set b
c
of experiments, we sought to determine whether ACO alters the
time course of IK(DR) induced by repetitive stimuli in these cells. 800
Under control conditions, a single 1-s depolarizing step to +50 mV
from a holding potential of −50 mV produced an exponential
decline with a time constant of 356 ± 9 ms (n = 7). However, the
400
time constant for 1-s repetitive pulses to +50 mV, each of which
lasted 30 ms with 20-ms interval at −50 mV between the depo-
0 200 400 600 800 1000
larizing pulses, was significantly reduced to 342 ± 8 ms (n = 7). As
Time (msec)
shown in Fig. 5, the results indicates a progressive increase in the
decline of IK(DR) in response to rapid depolarizing stimuli. During Fig. 5. Time course of excessive accumulative inactivation of IK(DR) in the absence
the exposure to ACO, the value of time constant obtained during and presence of ACO. Current amplitudes of IK(DR) were measured during repetitive
this train of short repetitive pulses was further reduced. Addition depolarizations from −50 to +50 mV with a duration of 30 ms at a rate of 20 Hz. a:
of 1 and 10 ␮M ACO decreased the time constants to 301 ± 7 ms control; b: 1 ␮M ACO; and c: 10 ␮M ACO. Smooth curves in a, b, c show the single-
exponential fits with time constants of 342, 301, and 227 ms, respectively. Notably,
(n = 6) and 227 ± 7 ms (n = 7), respectively. Therefore, there was
in addition to the inhibition of IK(DR) amplitude, ACO can increase the rate of exces-
an excessive accumulative inactivation of IK(DR) in the presence of sive accumulative inactivation of IK(DR) elicited by repetitive stimuli. Mean ± SEM
ACO. (n = 5–8).
16
1600
Current amplitude (pA)
1200
800
400
0 ing repetitive depolarizations. This major ion conductance present
-60 -40 -20 0 20
Membrane potential (mV)
Fig. 6. Effect of ACO on the averaged I–V relations of IK(DR) in Jurkat T-lymphocytes
treated with amiloride. Cells were preincubated with amiloride (30 ␮M) for 6 h. In
these experiments, amiloride-treated cells were bathed in Ca2+ -free Tyrode’s solu-
tion. In each cell examined, the IK(DR) obtained in the absence (䊉) and presence ()
of 10 ␮M ACO was elicited from −50 to different potentials which ranged from −50
to +50 mV with 10-mV increments (mean ± SEM; n = 5–7 for each point). Current
amplitude was measured at the end of each depolarizing pulse.
the magnitude of ACO-induced inhibition of IK(DR) between control
cells and cells treated with amiloride (Fig. 6). For example, when
the cells treated with amiloride (30 ␮M) were depolarized from
−50 to +30 mV, the IK(DR) amplitude measured at the end of the
depolarizing pulses was decreased by 72 ± 11% (n = 6) in the pres-
ence of 10 ␮M ACO. Under the same voltage protocol, ACO (10 ␮M)
decreased the current amplitude by 73 ± 10% (n = 7) measured from
control cells (i.e., in the absence of amiloride treatment). Similar
results were also obtained in cells which were preincubated with
tetrodotoxin (1 ␮M). The results thus led us to suggest that the
inhibitory effect of ACO on IK(DR) in these cells is not associated
with its effect on Na+ channels.
3.7. Effect of ACO on IK(DR) in RAW 264.7 macrophages
In a final set of experiments, we evaluated whether ACO has any
effect on IK(DR) in different types of immune cells. In these experi-
ments, RAW 264.7 cells were bathed in Ca2+ -free Tyrode’s solution
and the recording pipette was filled with K+ -containing solution.
A B
150
Current amplitude (pA)
100
50
0
-50
0 100
Time (msec)
Fig. 7. Inhibitory effect of ACO on IK(DR) in RAW 264.7 macrophages. In these experiments, cells were bathed in Ca2+ -free Tyrode’s solution and the recording pipette was
filled with K+ -containing solution. (A) Superimposed current traces of IK(DR) obtained in the absence (a) and presence (b) of 3 ␮M ACO. IK(DR) was elicited when the cell was
depolarized from −50 to +50 mV. (B) Summary of data depicting the effects of ACO (3 and 10 ␮M) on the amplitude of IK(DR) in RAW 264.7 cells (mean ± SEM, n = 5–7 for each
bar). Current amplitudes were measured at the end of each depolarizing pulse. *Significantly different from control.
S.-N. Wu et al. / Toxicology 289 (2011) 11–18
The IK(DR) was elicited when the cell was depolarized from −50 to
+50 mV. As illustrated in Fig. 7, the results showed that ACO at a
concentration of 3 and 10 ␮M was able to suppress the amplitude
of IK(DR) significantly in these cells; however it did not alter the
kinetics of IK(DR) activation and inactivation.
4. Discussion
The IK(DR) of Jurkat T-lymphocytes shown here can be observed
following the depolarizing pulses to −30 mV, were increased in
amplitude with more positive membrane potentials. The amplitude
of IK(DR) was inactivated as a result of prolonged depolarization (i.e.,
1 s). The amplitude of this current was also noted to inactivate dur-
in lymphocytes was thus identified as an outward K+ current sim-
40 60 ilar to KV 1.3-encoded current (Helms et al., 1997; Villalonga et al.,
2010). Addition of ACO to these cells was also noted to decrease the
amplitude of IK(DR) .
In our study, the effects of ACO on IK(DR) clearly were not lim-
ited to its inhibition of the current amplitude. Specifically, when
cells were exposed to ACO, the time course of IK(DR) inactivation
in response to sustained membrane depolarization became faster,
while no discernible change in the initial rising phase of current
was seen. This agent produced an increase in IK(DR) inactivation in
a concentration- and time-dependent manner. The results thus led
us to propose that blocking of IK(DR) by ACO inherently is not instan-
taneous, but develops with time after the channel becomes opened
and thereby produces an increase in current inactivation. In other
words, it tends to have a higher affinity for the open state in the
KV channel. As a result, the transition from opened to inactivated
state becomes faster during the exposure to ACO. It is thus possible
that ACO and its structurally related alkaloids binds to the C-type
inactivated state of the channel and/or blocks a prolonged channel
opening in KV 1.3 channels.
T lymphocytes were previously reported to express ␣7 -selective
nicotinic acetylcholine receptors (Razani-Boroujerdi et al., 2007; De
Rosa et al., 2009; Koval et al., 2009). ACO was shown to be an antago-
nist of ␣7 -nicotinic receptors (Hardick et al., 1995). One may expect
that the ACO-induced block of IK(DR) observed in Jurkat T lympho-
cytes is associated with its ability to bind to nicotinic receptors.
However, the inability of nicotine to reverse ACO-induced inhi-
bition of IK(DR) makes it unlikely that the binding to cholinergic
receptors in Jurkat T lymphocytes is responsible for its effect on
IK(DR) shown here.
a 120
b
Current amplitude (pA)
80 *
*
40
0
200 300 400
)
)
l
ro
μM
μM
nt
co
(3
0
(1
O
O
AC
AC
 S.-N. Wu et al. / Toxicology 289 (2011) 11–18
T-lymphocytes were demonstrated to express Na+ channels
sensitive to be inhibited by amiloride (Lai et al., 2000). Previous
studies have reported that ACO has a depressant action on Na+ cur-
rents in neuronal cells (Wu et al., 2008). However, in Jurkat T cells
treated with amiloride, ACO-induced inhibition of IK(DR) remained
unaltered. Therefore, the inhibition of IK(DR) caused by ACO in lym-
phocytes is direct and unlinked to its effect on Na+ current.
Recent studies showed that boiling of crude aconite roots
could decrease the amount of normal diterpene alkaloids due to
hydrolytic action and increase the concentration of lipo-alkaloids,
which might result in reduction of their toxicity (Borcsa et al., 2011).
However, when the ACO solution (10 ␮M) was boiled with a dura-
tion (at 100 ◦ C, 10 min), ACO was noted to exert little or no effects on
IK(DR) in Jurkat T cells (data not shown). Therefore, it is tempting to
anticipate that the structure of diterpene alkaloids is an important
site for the depressant action of ACO on IK(DR) in lymphocytes.
The ACO concentration used to produce anti-nociceptive or anti-
inflammatory effect appears to be close to its IC50 value for the
inhibition of IK(DR) (Oyama et al., 1994; Ameri, 1998; Ivy Carroll
et al., 2007). The IC50 value required for ACO-induced inhibition
of IK(DR) in Jurkat T-lymphocytes was 5.6 ␮M in this study. The
minimal reaction scheme presented here also prompted us to esti-
mate a KD value of 6.8 ␮M for its inhibition of IK(DR) in these
cells. Together with quantitative description of the time course of
IK(DR) inactivation measured at different ACO concentrations, our
results indicate that ACO can act as a state-dependent open-channel
blocker in Jurkat T lymphocytes. The IC50 value for anti-proliferative
properties against human tumor cells was reported to be around
10–20 ␮M (Chodoeva et al., 2005). Therefore, the effect of ACO
shown here may occur at a concentration achievable in humans
(Nakae et al., 2008; Yang et al., 2010). However, this compound
did not alter the inactivation kinetics of IK(DR) in RAW 264.7
macrophages.
KV 1.3 blockers have been shown to ameliorate adoptive experi-
mental autoimmune encephalomyelitis induced by myelin-specific
memory T cells, a model for multiple sclerosis (Vennekamp et al.,
2004; Gilhar et al., 2011), and to prevent inflammatory bone resorp-
tion in experimental periodontal disease caused mainly by memory
cells (Valverde et al., 2005). Regardless of the mechanism of its
action, there is a relevant link between the effects of ACO on
immune cells and its inhibitory effect on the KV 1.3 channel (Chandy
et al., 2004; Panyi, 2005). The inhibition by ACO of KV 1.3 channel
activity is likely to affect KV 1.3 channel-related immunomodula-
tion in the immune system. ACO-induced blockade of IK(DR) and
its binding to acetylcholine receptors may synergistically affect the
functional activity of immune cells like Jurkat T-lymphocytes. It is
thus necessary to evaluate the appropriate use of ACO, as this com-
pound has a narrow therapeutic index and some adverse effects
may occur in vivo.
Conflict of interest
The authors declare no competing interests in this work.
Acknowledgements
The authors would like to thank Hsung Peng, Tai-I Hsu and Pei-
Yu Wu for their helpful assistances in cell culture. The work in
this laboratory was partly supported by a grant from the National
Science Council (NSC-98-2320-B-006-MY3).
References
Ameri, A., 1998. The effects of Aconitum alkaloids on the central nervous system.
Prog. Neurobiol. 56, 211–235.
17
Beeton, C., Pennington, M.W., Wulff, H., Singh, S., Nugent, D., Crossley, G., Khaytin,
I., Calabresi, P.A., Chen, C.Y., Gutman, G.A., Chandy, K.G., 2005. Targeting effector
memory T cells with a selective peptide inhibitor of Kv 1.3 channels for therapy
of autoimmune diseases. Mol. Pharmacol. 67, 1369–1381.
Borcsa, B., Csupor, D., Forgo, P., Widowitz, U., Bauer, R., Hohmann, J., 2011. Aconitum
lipo-alkaloids—semisynthetic products of the traditional medicine. Nat. Prod.
Commun. 6, 527–536.
Chandy, K.G., Pennington, M., Knaus, H.-G., Wulff, H., 2004. K+ channel expression
during B cell differentiation: implications for immunomodulation and autoim-
munity. J. Immunol. 173, 776–786.
Chodoeva, A., Bosc, J.J., Guillon, J., Descendit, A., Petraud, M., Absalon, C., Vitry, C.,
Jarry, C., Robert, J., 2005. 8-O-Azeloyl-14-benzoylaconine: a new alkaloid from
the roots of Aconitum karacolicum Rapcs and its antiproliferative activities.
Bioorg. Med. Chem. 13, 6493–6501.
Chung, I., Schlichter, L.C., 1997. Native Kv 1.3 channels are upregulated by protein
kinase C. J. Membr. Biol. 156, 73–85.
Dasyukevich, O.I., Solyanik, G.I., 2007. Comparative study of anticancer efficacy of
aconitine-containing agent BC1 against ascite and solid tumors of Ehrlich’s car-
cinoma. Exp. Oncol. 29, 317–319.
De Rosa, M.J., Dionisio, L., Agriello, E., Bouzat, C., Esandi Mdel, C., 2009. Alpha 7
nicotinic acetylcholine receptor modulates lymphocyte activation. Life Sci. 85,
444–449.
Garcia-Calvo, M., Leonard, R.J., Novick, J., Stevens, S.P., Schmalhofer, W., Kac-
zorowski, G.J., Garcia, M.L., 1993. Purification, characterization, and biosynthesis
of margatoxin, a component of Centruroides margaritatus venom that selec-
tively inhibits voltage-dependent potassium channels. J. Biol. Chem. 268,
18866–188874.
Garmanchouk, L.V., Pyaskovskaya, O.N., Yanish, Y.V., Shlyakhovenko, V.A., Dasyuke-
vich, O.I., Solyanik, G.I., 2005. Influence of aconitine-containing herbal extract
BC-1 proliferative and electrokinetic characteristics of endothelial cells. Exp.
Oncol. 27, 262–266.
Gilhar, A., Bergman, R., Assay, B., Ullmann, Y., Etzioni, A., 2011. The beneficial effect
of blocking Kv 1.3 in the psoriasiform SCID mouse model. J. Invest. Derm. 131,
118–124.
Hardick, D.J., Cooper, G., Scott-Ward, T., Blagbrough, I.S., Potter, B.V., Wonnacott,
S., 1995. Conversion of the sodium channel activator aconitine into a potent
␣7-selective nicotinic ligand. FEBS Lett. 365, 79–82.
Helms, L.M., Felix, J.P., Bugianesi, R.M., Garcia, M.L., Stevens, S., Leonard, R.J., Knaus,
H.G., Koch, R., Wanner, S.G., Kaczorowski, G., Slaughter, R.S., 1997. Margatoxin
binds to a homomultimer of KV 1.3 channels in Jurkat cells. Comparison with
KV 1.3 expressed in CHO cells. Biochemistry 36, 3737–3744.
Huang, M.H., Shen, A.Y., Wang, T.S., Wu, H.M., Kang, Y.F., Chen, C.T., Hsu, T.I., Chen,
B.S., Wu, S.N., 2011. Inhibitory action of methadone and its metabolites on erg-
mediated K+ current in GH3 pituitary tumor cells. Toxicology 280, 1–9.
Ivy Carroll, F., Ma, W., Navarro, H.A., Abraham, P., Wolckenhauer, S.A., Damai, M.I.,
Martin, B.R., 2007. Synthesis, nicotinic acetylcholine receptor binding, antinoci-
ceptive and seizure properties of methyllycaconitine analogs. Bioorg. Med.
Chem. 15, 678–685.
Kemmer, G., Keller, S., 2010. Nonlinear least-squares data fitting in Excel spread-
sheets. Nat. Protocol 5, 267–281.
Kostenko, S., Khan, M.T., Sylte, I., Moens, U., 2011. The diterpenoid alkaloid norox-
oaconitine is a Mapkap kinase-5 (MK5/PRAK) inhibitor. Cell Mol. Life Sci. 68,
289–301.
Koval, L.M., Yu Lykhmus, O., Omelchenko, D.M., Komisarenko, S.V., Skok, M.V., 2009.
The role of alpha7 nicotinic acetylcholine receptors in B lymphocyte activation.
Ukr. Biokhim. Zh. 81, 5–11.
Lai, Z.F., Chen, Y.Z., Nishimura, Y., Nishi, K., 2000. An amiloride-sensitive and voltage-
dependent Na+ channel in an HLA-DR-restricted human T cell clone. J. Immunol.
165, 83–90.
Lin, A.A., Wang, Y.J., Lin, M.W., Wu, S.N., 2007. Differential mechanisms of
margatoxin- and quercetin-induced regulation of voltage-gated ion currents in
Jurkat T lymphocytes. FASEB J. 21, A957.
Lin, M.W., Wang, Y.J., Liu, S.I., Lin, A.A., Lo, Y.C., Wu, S.N., 2008. Characterization of
aconitine-induced block of delayed rectifier K+ current in differentiated NG108-
15 neuronal cells. Neuropharmacology 54, 912–923.
Makino, T., Kato, K., Mizukami, H., 2009. Processed aconite root prevents cold-stress-
induced hypothermia and immuno-suppression in mice. Biol. Pharm. Bull. 32,
1741–1748.
Maldonado, D., Schumann, M., Nghiem, P., Dong, Y., Gardner, P., 1991. Prostaglandin
E1 activates a chloride current in Jurkat T lymphocytes via cAMP-dependent
protein kinase. FASEB J. 5, 2965–2970.
Marom, S., Goldstein, S.A., Kupper, J., Levitan, L.B., 1993. Mechanism and modulation
of inactivation of the Kv3 potassium channels. Recept. Channels 1, 81–88.
Matteson, D.R., Deutsch, C., 1984. K channels in T lymphocytes: a patch-clamp study
using monoclonal antibody adhesion. Nature 307, 468–471.
Mogg, A.J., Whiteaker, P., Mcintosh, J.M., Marks, M., Collins, A.C., Wonnacott, S., 2002.
Methyllycaconitine is a potent antagonist of ␣-conotoxin-MII-sensitive presy-
naptic nicotinic acetylcholine receptors in rat striatum. J. Pharmacol. Exp. Ther.
302, 197–204.
Nakae, H., Fujita, Y., Igarashi, T., Tajimi, K., Endo, S., 2008. Serum aconitine con-
centrations after taking powdered processed Aconiti tuber. Biomed. Res. 29,
225–231.
Oyama, T., Isono, T., Suzuki, Y., Hayakawa, Y., 1994. Anti-nociceptive effects of
aconite tuber and its alkaloids. Am. J. Chin. Med. 22, 175–182.
Pang, B., Zheng, H., Shin, D.H., Jung, K.C., Ko, J.H., Lee, K.Y., Kang, T.M., Kim,
S.J., 2010. TNF-a inhibits the CD3-mediated upregulation of voltage-gated
18 S.-N. Wu et al. / Toxicology 289 (2011) 11–18
K+ channel (KV 1.3) in human T cells. Biochem. Biophys. Res. Commun. 391,
909–914.
Panyi, G., 2005. Biophysical and pharmacological aspects of K+ channels in T lym-
phocytes. Eur. Biophys. J. 34, 515–529.
Pegoraro, S., Lang, M., Dreker, T., Kraus, J., Hamm, S., Meere, C., Feurle, J., Tasler,
S., Prütting, S., Kuras, Z., Visan, V., Grissmer, S., 2009. Inhibitors of potassium
channels KV 1.3 and IK-1 as immunosuppressants. Bioorgan. Med. Chem. Lett.
19, 2299–2304.
Razani-Boroujerdi, S., Boyd, R.T., Davila-Garcia, M.I., Nandi, J.S., Mishra, N.C., Singh,
S.P., Pena-Philippides, J.C., Langley, R., Sopori, M.L., 2007. T cells express ␣7-
nicotinic acetylcholine receptor subunits that require a functional TCR and
leukocyte-specific protein tyrosine kinase for nicotine-induced Ca2+ response.
J. Immunol. 179, 2889–2898.
Shen, A.Y., Tsai, J.H., Teng, H.C., Huang, M.H., Wu, S.N., 2007. Inhibition of
intermediate-conductance Ca2+ -activated K+ channel and cytoprotective prop-
erties of 4-piperidinomethyl-2-isopropyl-5-methlphenol. J. Pharm. Pharmacol.
59, 679–685.
Singhuber, J., Zhu, M., Prinz, S., Kopp, B., 2009. Aconitum in traditional chinese
medicine—a valuable drug or an unpredictable risk? J. Ethnopharmacol. 126,
18–30.
Tang, H.Y., Han, Y.M., Yan, D., Zhang, S.F., Luo, Y., Xiao, X.H., Liu, R.H., Peng, C., 2010.
Microcalorimetric investigation of toxic effects of diester diterpenoid alkaloids
from Aconiti class on Tetrahymena thermophila BF5 growth. Acta Chim. Sin. 68,
205–210.
Valverde, P., Kawai, T., Taubman, M.A., 2005. Potassium channel-blockers as thera-
peutic agents to interfere with bone resorption of periondontal disease. J. Dent.
Res. 84, 488–499.
Vennekamp, J., Wulff, H., Beeton, C., Calabresi, P.A., Grissmer, S., Hänsel, W.,
Chandy, K.G., 2004. Kv 1.3-blocking 5-phenylakoxypsoralens: a new class of
immunomodulators. Mol. Pharmacol. 65, 1364–1374.
Villalonga, N., David, M., Bielańska, J., González, T., Parra, D., Soler, C., Comes, N.,
Valenzuela, C., Felipe, A., 2010. Immunomodulatory effects of diclofenac in
leukocytes through the targeting of Kv 1.3 voltage-dependent potassium chan-
nels. Biochem. Pharmacol. 80, 858–866.
Wu, S.N., Chen, B.S., Lin, M.W., Liu, Y.C., 2008. Contribution of slowly inacti-
vating potassium current to delayed firing of action potentials in NG108-15
neuronal cells: experimental and theoretical studies. J. Theor. Biol. 252,
711–721.
Wu, S.N., Wu, P.Y., Tsai, M.L., 2011. Characterization of TRPM8-like channels acti-
vated by the cooling agent icilin in the macrophage cell line RAW 264.7. J. Membr.
Biol. 241, 11–20.
Wu, S.N., Yu, H.S., Jan, C.R., Li, H.F., Yu, C.L., 1998. Inhibitory effects of berberine on
voltage- and calcium-activated potassium currents in human myeloma cells.
Life Sci. 62, 2283–2294.
Xu, N., Zhao, D.F., Liang, X.M., Zhang, H., Xiao, Y.S., 2011. Identification of diter-
penoid alkaloids from the roots of Aconitum kusnezoffii Reihcb. Molecules 16,
3345–3350.
Yan, Z.C., Chen, D., Wu, X.Z., Xie, G.R., Ba, Y., Yan, Z., 2007. Effects of aqueous extracts
of Aconitum carmichaeli, Rhizoma bolbostemmatis, Phytolacca acinosa, Panax noto-
ginseng and Gekko swinhonis Guenther on Bel-7402 cells. World J. Gastroenterol.
13, 2743–2746.
Yang, Y., Chen, J., Shi, Y.P., 2010. Determination of aconitine, hypaconitine and
mesaconitine in urine using hollow fiber liquid-phase microextraction com-
bined with high-performance liquid-chromatography. J. Chromatogr. B: Anal.
Technol. Biomed. Life Sci. 878, 2811–2816.
Zhang, M., Long, Y., Sun, Y., Wang, Y., Li, Q., Wu, H., Guo, Z., Li, Y., Niu, Y., Li, C.,
Liu, L., Mei, Q., 2011. Evidence for the complementary and synergistic effects of
the three-alkaloid combination regimen containing berberine, hypaconitine and
skimmianine on the ulcerative colitis rats induced by trinitrobenzene-sulfonic
acid. Eur. J. Pharmacol. 651, 187–196.