atopic dermatitis ( ad ) is an inflammatory , chronically relapsing , non - contagious and pruritic skin disorder characterized by allergic symptoms such as redness , inflammation , and itching . more studies of anti - atopic agents must be conducted because the exact pathogenesis of ad is unknown and a definitive treatment is lacking . taxifolin 3-o--d - glucopyranoside ( taxifolin glycoside ) is a flavonoid compound isolated from rhododendron mucronulatum , which has been used for treatment of rheumarthritis , neuralgia , and inflammation traditional oriental medicine . recent studies indicate that taxifolin glycoside inhibits dendritic cell - mediated immune response and demonstrates anti - inflammatory activity in nc / nga mice : an atopic dermatitis model . in a therapeutic efficacy study in nc / nga mice with ad - like lesions , a rapid and profound restoration of skin barrier function was observed in the group treated with the taxifolin glycoside - loaded pep-1 peptide - conjugated elastic liposomes ( pep1-el ) formulation . the k channel controlled by the human ether - a - go - go related gene ( herg ) conducts the rapidly activating delayed rectifier k current ( ikr ) in cardiac myocytes , which plays a central role in the repolarization of action potentials [ 1 - 3 ] . loss of function or blockade of the herg k channel can induce long qt syndrome ( lqts ) , which is characterized by a prolonged qt interval on an electrocardiogram and can result in fatal arrhythmias such as torsade de pointes . many drugs have reported side effects , including herg k current inhibition , which can lead to acquired lqts [ 9 - 12 ] . however , little information is available regarding the anti - arrhythmic effect and herg k channel blockade that are caused by taxifolin glycoside . in this study , we tested the cardiovascular safety of taxifolin glycoside by exmining its effects on herg k channels using a whole cell patch clamp method . ( professor , department of physiology , college of medicine , sungkyunkwan university , suwon , korea ) . herg cdna was subcloned into mammalian expression vector pcdna3.1 ( invitrogen ) for transfection into cells . transfection of herg into chinese hamster ovary ( cho ) cells was achieved via the lipofectamin method ( life technologies ) . the stably transfected herg - cho cells were generated through g418 ( gibco ) antibiotic selection . the transfected clones were selected through the use of 1,000 g / ml of g418 . the cho cells stably expressed herg ( herg - cho ) were maintained in f-12 supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) and 500 g / ml g418 . for electrophysological recordings , the cells were harvested from the culture dish via trypsinization and were seeded onto a glass coverslip . ( professor , college of pharmacy , chung - ang university , seoul , korea ) and was dissolved in distilled water to obtain a 500 mm stock solution , which was stored at -20. taxifolin glycoside was diluted in external solution to achieve the final concentration . cells on a coverslip were transferred to a recording chamber mounted on the stage of an inverted microscope ( olympus ) . for the herg k current recording , the bath was perfused with hepes - buffered tyrode solution containing ( in mm ) nacl 155 , kcl 5 , mgcl2 1 , hepes 10 , and glucose 10 ( ph 7.3 with naoh ) . the pipette solution contained ( in mm ) kcl 140 , mg - atp 5 , mgcl2 1 , kh2po4 10 , egta 0.1 , and hepes 5 ( ph 7.2 with koh ) . the electrodes were constructed from borosilicate glass ( wpi ) using a micropipette puller ( narishige ) and were heat - polished with a microforge ( narishige ) . the resistance of the electrode when filled with the pipette solution was 2~3 m. membrane currents were recorded with an axopatch 200b amplifier ( axon instruments ) and a digidata 1322a ( axon instruments ) . the pclamp 9.2 software ( axon instruments ) was used to generate voltage clamp protocols , acquire data , and analyze current traces . data analysis and curve fitting were performed with clampfit 9.2 ( axon instruments ) and origin 8.0 software ( originlab ) . ( professor , department of physiology , college of medicine , sungkyunkwan university , suwon , korea ) . herg cdna was subcloned into mammalian expression vector pcdna3.1 ( invitrogen ) for transfection into cells . transfection of herg into chinese hamster ovary ( cho ) cells was achieved via the lipofectamin method ( life technologies ) . the stably transfected herg - cho cells were generated through g418 ( gibco ) antibiotic selection . the transfected clones were selected through the use of 1,000 g / ml of g418 . the cho cells stably expressed herg ( herg - cho ) were maintained in f-12 supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) and 500 g / ml g418 . for electrophysological recordings , the cells were harvested from the culture dish via trypsinization and were seeded onto a glass coverslip . ( professor , college of pharmacy , chung - ang university , seoul , korea ) and was dissolved in distilled water to obtain a 500 mm stock solution , which was stored at -20. taxifolin glycoside was diluted in external solution to achieve the final concentration . cells on a coverslip were transferred to a recording chamber mounted on the stage of an inverted microscope ( olympus ) . for the herg k current recording , the bath was perfused with hepes - buffered tyrode solution containing ( in mm ) nacl 155 , kcl 5 , mgcl2 1 , hepes 10 , and glucose 10 ( ph 7.3 with naoh ) . the pipette solution contained ( in mm ) kcl 140 , mg - atp 5 , mgcl2 1 , kh2po4 10 , egta 0.1 , and hepes 5 ( ph 7.2 with koh ) . the electrodes were constructed from borosilicate glass ( wpi ) using a micropipette puller ( narishige ) and were heat - polished with a microforge ( narishige ) . the resistance of the electrode when filled with the pipette solution was 2~3 m. membrane currents were recorded with an axopatch 200b amplifier ( axon instruments ) and a digidata 1322a ( axon instruments ) . the pclamp 9.2 software ( axon instruments ) was used to generate voltage clamp protocols , acquire data , and analyze current traces . data were expressed as meanstandard error of the mean ( sem ) . data analysis and curve fitting were performed with clampfit 9.2 ( axon instruments ) and origin 8.0 software ( originlab ) . we investigated the effect of taxifolin glycoside on herg k current stably expressed in cho cells using the whole - cell patch clamp technique . the herg k currents were elicited from a holding potential of -80 mv by the test pulses ranging from -60 mv to + 40 mv in 10 mv steps ( istep ) . each test pulse had 4 seconds duration and was followed by a repolarization step to -50 mv for 2 seconds to evoke tail current ( itail ) . the herg k currents were first recorded under control conditions , and then 30 m taxifolin glycoside was added into the bath for 20 minutes . taxifolin glycoside reduced tail currents of herg k channels during the repolarizing pulse ( fig . normalized current - voltage ( i - v ) relationships of itail are illustrated in fig . 1b ( n=5 ) , which shows that itail was increased with depolarizing voltages and reached a plateau at approximately + 30 mv . taxifolin glycoside reduced itail amplitudes after a test pulse to 0 mv . at taxifolin glycoside concentrations of 5 , 10 , 30 , 50 and 100 m , the peak amplitude of the itail were measured in the same cell and were normalized to the control value . taxifolin glycoside blocked itail in a concentration - dependent manner . to obtain the half maximal effective concentration ( ec50 ) of taxifolin glycoside the ec50 of taxifolin glycoside was 9.60.7 m ( n=5 , fig . 1c ) . the fractional blockages in itail were 12.02.5% , 18.14.7% , 26.34.5% , 35.45.6% and 36.93.0% in the 5 , 10 , 30 , 50 and 100 m taxifolin glycoside groups , respectively ( n=5 ) . the effect of taxifolin glycoside was irreversible ( data not shown ) . to further understand the inhibitory effect of taxifolin glycoside on herg k channels , we investigated the changes in channel kinetics induced by taxifoline glycoside . we first examined the effects of 30 m taxifolin glycoside on the time constants of activation and the activation curve . to obtain the time constant of activation , the increasing phase of istep in fig . 1a was fitted to a single exponential function for test potentials in the range of -30 mv to + 20 mv . the time constant of activation was significantly smaller in the presence of 30 m taxifolin glycoside and their time constants were reduced by 29.94.3% at + 10 mv ( n=5 , p<0.05 , fig . relative to the control , herg k currents after taxifolin glycoside treatment activated more rapidly . and the normalized data were plotted against the voltage and fitted with the boltzmann function ( fig . taxifolin glycoside significantly changed the half - maximal activation voltages ( v1/2 : 2.72.1 mv and -2.20.1 mv ; in the control group and in the group treated with 30 m taxifolin glycoside , respectively ; n=5 , p<0.05 ) . a 30 m concentration of taxifolin glycoside produced a significant negative shift of the activation curve . we next examined the effects of taxifolin glycoside on the time constants of steady - state inactivation , onset of inactivation and recovery from inactivation . to measure steady - state inactivation , a special protocol was used to inactivate the channel at a holding potential of + 20 mv for 2 seconds , recover the channel from inactivation at various potentials ranging from -120 mv to + 60 mv for 15 seconds in 10 mv steps , and then measure the resulting peak outward current at a constant + 20 mv in the absence and the presence of 30 m taxifolin glycoside ( fig . 3b , the inactivation outward current amplitude was normalized and plotted against the test pulse potential , producing the steady - state inactivation curve . taxifolin glycoside did not affect the half - maximal inactivation values of herg k channels ( v1/2 : -43.35.4 mv and -37.87.4 mv ; in control and after treatment with 30 m taxifolin glycoside , respectively ; n=5 ) . the time constant of channel inactivation was also assessed . to obtain the time constants of steady - state inactivation , the decays of the outward current in fig . although taxifolin glycoside caused a slight decrease in the time constants of direct channel inactivation , the changes were not statistically significant ( n=5 , p>0.05 ) . the channels were first inactivated by clamping the membrane at + 20 mv for 2 seconds , followed by a prepulse to -120 mv for 15 microseconds . this prepulse was sufficiently long to allow for the rapid recovery of channels from inactivation , but short enough to prevent significant channel deactivation . following the recovery prepulse , a series of test pulses were delivered with potentials ranging from -80 mv to + 60 mv , resulting in large , outward inactivating currents . the current traces at the onset of inactivation in the absence and the presence of 30 m taxifolin glycoside are shown in fig . 4a . the time constants for the onset of inactivation were obtained by fitting a single exponential function to the decaying current traces during the third pulse of the protocol . the time constants of onset of inactivation were significantly decreased in the presence of 30 m taxifolin glycoside ( n=5 , p<0.05 , fig . the fully activated i - v protocol was used to determine the recovery from inactivation . each current was obtained through depolarization to + 20 mv for 2 seconds to inactivate the herg k channels , followed by varying the repolarizing pulses to test potentials between -120 mv and + 30 mv in 10 mv steps . the prepulse potential at + 20 mv was sufficiently positive to induce full conductance of the herg k channels but also rendered a large number of the channels inactive . the current traces for recovery from inactivation in the absence and the presence of 30 m taxifolin glycoside are shown in fig . 5a . the increasing phase of the tail current represents the rapid recovery from inactivated states to open states . the time constant for recovery from inactivation were obtained by fitting a single exponential function to the initial increase in tail current amplitude ( n=5 , fig . the present study demonstrated that taxifolin glycoside directly blocks herg k channels in herg - cho cells . taxifolin glycoside induced 2 distinct changes in herg k channel gating : ( 1 ) facilitating activation ( negative shift of the voltage - dependence of activation and accelerated activation time constant ) and ( 2 ) facilitating of inactivation ( reduced onset of inactivation time constant ) . in fig . 1 , the blockage by taxifolin glycoside was significantly increased at + 30 mv at which point the herg k channel was fully activated . these results suggest that blockage by taxifolin glycoside might occur via open - state blockage . taxifolin glycoside produced a significant negative shift of the activation curve , and the time constant of activation was significantly decreased in the presence of taxifolin glycoside ( fig . these data indicate that the activation of herg k channels was accelerated by taxifolin glycoside and that taxifolin glycoside may block the herg k channel when it is open . 4 ) , although the properties of steady - state inactivation and recovery from inactivation were not changed ( fig . 3 and fig . 5 ) . these results indicate that the onset of inactivation of herg k channels was accelerated by taxifolin glycoside and that taxifolin glycoside may also exhibit affinity for the inactivated state . because herg k channel blockade has the potential to cause extensive side effects , in addition to lqts and arrhythmias , in the process of developing new pharmaceutical drugs , the test for drug - induced herg k channel blockade should be conducted prior to initiating in vivo toxicology studies . therefore , an understanding of drug - herg k channel interaction at the molecular level is necessary for rational drug design . with regard to the relationship among herg k current blockade , qt prolongation and fatal arrhythmia , redfern et al . recommended a 30-fold safety margin between the maximum effective therapeutic plasma concentration of a drug and the ic50 of herg . in this study , we found that taxifolin glycoside directly inhibits herg k currents in a concentration - dependent manner , with an ec50 of 9.60.7 m . however , taxifolin glycoside of high concentration ( more than 100 m ) did not produce further decrease of herg k currents . 1c shows an unusual situation where the inhibition curve plateaus well above the control values . in this case , ec50 refers to relative ic50 which define the half concentration between the top and bottom plateaus of the curve . based on this data , recently , taxifolin glycoside has demonstrated anti - inflammatory and immune modulatory effects at concentrations between 10 and 20 m in in vitro experiments [ 4 - 6 ] . however , the concentrations that are effective in vitro are higher than the recommended therapeutic plasma concentrations . taxifolin glycoside also has the possibility to induce a cardiac arrhythmia , although the maximum inhibitory effects on herg k currents was 36.93.0% , and taxifolin glycoside concentrations 100 m did not produce a further dcrease of herg k currents . since outward k currents play an important role during plateau repolarization and in determining the configuration of the action potential , small changes in conductance can significantly alter the effective refractory period , hence the action potential duration . so , if we use taxifolin glycoside as a topical ointment to treat inflammatory skin lesions such as atopic dermatitis and contact dermatitis , such treatment would be unlikely to produce side effects because the transcutaneous absorption rate is 1% that of oral delivery or injection . in conclusion , taxifolin glycoside inhibited herg k currents that function by facilitating activation and inactivation process . our results provide an understanding of the cardiovascular risk profile of the new drug taxifolin glycoside .
taxifolin glycoside is a new drug candidate for the treatment of atopic dermatitis ( ad ) . 
 many drugs cause side effects such as long qt syndrome by blocking the human ether - a - go - go related gene ( herg ) k+ channels . to determine whether taxifolin glycoside would block herg k+ channels , we recorded herg k+ currents using a whole - cell patch clamp technique . 
 we found that taxifolin glycoside directly blocked herg k+ current in a concentration - dependent manner ( ec50=9.60.7 m ) . 
 the activation curve of herg k+ channels was negatively shifted by taxifolin glycoside . 
 in addition , taxifolin glycoside accelerated the activation time constant and reduced the onset of the inactivation time constant . 
 these results suggest that taxifolin glycoside blocks herg k+ channels that function by facilitating activation and inactivation process .