Page 17 - I. Chemical biology and drug discovery

Sophora flavescens extracts elicit therapeutic effects on overactive bladder syndrome by activating
the large-conductance calcium-activated potassium channel
Heeji Jo¹, Jisun Han¹, Narasaem Lee¹, Hyun Jun Lee², Sung Joo Jang³, Seunghwan Ryu³, Sang Hyun Moh³, Jae Hoon Cheong², and Chul-Seung Park¹
¹School of Life Sciences and Integrated Institute of Biomedical Research, Gwangju Institute of Science and Technology, Gwangju 61005, South Korea,
²Department of Pharmacy, Sahmyook University, Seoul 01795, South Korea,
³Anti-Aging Research Institute, BIO-FD&C Co., Ltd, Incheon 21990, South Korea
BACKGROUND AIM

The large-conductance calcium-activated potassium channel (BK Ca channel), also called Maxi-K or Slo1, is a family of K + channels activated by membrane depolarization Herein, we wanted to validate the efficacy of Kushen
and an increase of intracellular Ca 2+ concentration. When the channels open, potassium ions flow out rapidly and membrane hyperpolarization is induced. This channel is crude extracts on BK Ca channel activity. We
related to various physiological processes including neural excitability, the release of hormone and neurotransmitter, innate immunity, and smooth muscle tone. In particular, optimized the extraction method using ethanol
it plays critical roles in the maintenance of the resting membrane potential and the phasic relaxation in urinary bladder smooth muscle (UBSM). (EtOH) fraction to increase the content of flavonoids
When the BK Ca channel expression level was down-regulated, the UBSM appeared a hyper-contractility which is called overactive bladder (OAB). OAB patients are living presenting the potentiation effect on BK Ca channel.
under constant stress due to their sensitive bladder that contracts frequently before filling up with urine. Meanwhile, activation of the BK Ca channel decreased UBSM We wanted to compare the effects of Kushen
excitability in guinea pigs by an endogenous ligand, testosterone. For these reasons, the BK Ca channel has emerged as a novel therapeutic target against OAB and its extracts on BK Ca channel activity to those of
chemical activators have been reported actively. In our previous report, several flavonoids, a class of plant metabolites, isolated from the roots of Sophora flavescens Aiton kurarinone, the major BK Ca channel activator among
(Fabaceae) significantly increased the activity of the BK Ca channel. Kushen flavonoids, in vitro cell-based assay. The
S. flavescens Ait. is a medicinal plant widely founded in Northeast Asia. Its root (Kushen, 苦参) has been used as a traditional Chinese herbal medicine for over a thousand mechanism of channel potentiation was analyzed
years to relieve fever and skin-itching. So far, 181 bioactive compounds have been identified from Kushen, among them, alkaloids and flavonoids are the major two chemical based on the patch-clamp recording of BK Ca
groups. Moreover, recent studies reported that the bioactive compounds induce estrogenic, anti-tumor and hepatoprotective effects. Although the novel therapeutic effects, channel macroscopic current. Furthermore, the
Kushen has the challenge to be utilized as a medicine for the specified use due to its complex composition. The bioactive compounds need lots of costs to be isolated with effects on the urinary activity of the OAB rat model
high purity or synthesized due to its structural complexity. In addition to the difficulty of quality control, Kushen has potential toxicity in rabbits, frogs, and mice at an excessive were verified.
dosage induced by some toxic alkaloid components.
METHODS

• Kushen extracts obtatined by 10% and 30% EtOH-water mixture for 5 h at 75 ℃ after water extraction to remove alkaloid components
• A cell-based assay using hyperactive BK Ca channel (G803D/N806K) with FluxOR potassium channel assay.
• Electrophysiological current recording based on Xenopus laevis oocyte expression system.
• Behavioral observation of SHR (hypertensive OAB rat model) after oral administration of Kushen extracts.
RESULTS

Figure 1. Effects of Kushen extracts on BK Ca Figure 2. Effects of Kushen extracts on BK Ca channel macroscopic currents
channel activity in a cell-based fluorescence A. Representative BK Ca channel current traces upon
assay treatment of 10% EtOH extract. B. Representative
BK Ca channel current traces upon treatment of 30%
EtOH extract. Kushen extracts were perfused from the
extracellular side with 3 µM of intracellular Ca 2+
concentration. Ionic currents were elicited with 100-ms
voltage step pulses from -80 to 180 mV with -100 mV
of holding voltage. The gray-colored line is showing
ionic current at 140 mV pulse. C. Conductance-
Voltage (G-V) relationship curve from panel A and B.
Conductance (G) was obtained from the peak tail
currents were normalized by the maximum
conductance of vehicle group (G o max ). The Boltzmann
equation (G = {(G max – G min ) / (1 + exp[(V 1/2 - V) / k])} +
G min ) was used for data fitting with Origin 9.1. k is
RT/zF, where R is the gas constant, T is temperature,
F is the Faraday constant, and z is gating charge.
Symbols are as follows: □, vehicle (n = 6); 10% EtOH
extracts - ●, 10 µg/mL (n = 4); ▼, 50 µg/mL (n = 6);
and 30% EtOH extracts - half-filled ●, 10 µg/mL (n =
6); half-filled ▼, 50 µg/mL (n = 7). D. V 1/2 changes
according to treatment of Kushen extracts. E. G /
G o max changes at 140-mV pulse according to treatment
of Kushen extracts.
Figure 3. Effects of Kushen extracts on time Figure 4. Effects of Kushen extracts on the voiding
A. Representative traces showing the change in fluorescence (RFU) constant of BK Ca channel gating behavior of OAB rats
over time upon treatment with 50 µg/mL of 10% and 30% EtOH
extracts. Kurarinone was used as a positive control at 3, 5, and 10 B
µM. Tl + -containing stimulus buffer was added after obtaining the
baseline for 20 s. B. Fold increase of RFU at 80 s after treating
stimulus buffer from panel A. C. Representative traces showing the
change in fluorescence (in RFU) over time upon treatment with
various concentrations of 10% and 30% EtOH extracts. D. Fold
increase of RFU at 80 s after treating stimulus buffer from panel C.
Symbols are as follows: □, vehicle; Kurarinone - gray-colored ◄, 3
µM; ►, 5µM; pentagon, 10 µM; 10% EtOH extract - ●, 10 µg/mL;
▲, 30 µg/mL; ▼, 50 µg/mL; ◆, 100 µg/mL; and 30% EtOH extract -
half-filled ●, 10 µg/mL; ▲, 30 µg/mL; ▼, 50 µg/mL; ◆, 100 µg/mL
(n = 4 for each group). Each dot or bar represents mean ± S.E.M. *
p < 0.05, ** p < 0.01, *** p < 0.001 compared to vehicle group. A. Effect of Kushen extracts at 50 µg/mL on activation time constant (τ activation )
and deactivation time constant (τ deactivation )(B). The outward current and the A. Number of urination for 3 h upon administration of Kushen extracts on
tail current were analyzed for τ deactivation and τ deactivation . The τ values were rats. B. Urine volume for 3 h upon administration of Kushen extracts on
obtained from fitting every independent data set using the double exponential rats. Kushen extracts were orally-administrated at single treatment for
function (y(t) = A fast exp(- t / τ fast ) + A slow exp(- t / τ slow ) + C) with Origin 9.1. different dosages, 25, 50, 100, and 200 mg/kg. Kurarinone was used as
Symbols are as follows: □, vehicle; 10% EtOH extracts - ▼, 50 µg/mL; and positive control at 2.5, 5 and 10 mg/kg (n = 6 ~ 12). Each bar represents
30% EtOH extracts – half-filled ▼, 50 µg/mL (n = 6 for each group). Each dot mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to SHR
represents mean ± S.E.M. * p < 0.05, ** p < 0.01 compared to vehicle group. saline group.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
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Lab Hompage address : http://life.gist.ac.kr/mnl/