Extracts Of Crinum Asiaticum Gained Activation Of KCNQ Potassium Channel And Potential Antiepileptic Effect

Part one:The preparation of extracts from Crinum Asiaticum and the quantitative determination of lycorineObjective:To prepare the extracts of Crinum Asiaticum and measure the contents of the main alkaloids.Methods:1 Preparation of the extracts from Crinum Asiaticum:After the dried bulb of Crinum asiaticum was crushed,the powder was extracted with 95%ethanol,and then the diacolation liquid was concentrated by reducing pressure.The residue material was leached by EtoAc,CH₂Cl₂ and Petro respectively.The part of ethyl acetate extraction,evaporated the solvent,and used for the study of pharmacodynamics and mechanism.2 The determination of the lycorine alkali content:High perfor-mance liquid chromatography（HPLC）,with Phenomenex C18 column,was applied in isocratic elution method,and the mobile phase was water and 1%trieth-ylamine methanol solution.Results:The solid extracts weight 1.6 g in the part of ethyl acetate extraction was separated from the raw dried Crinum Asiaticum weight 1.01 kg,and quantitative determination of lycorine was 6.0%containing in the solid extracts.Summary:The quantification amount of lycorine was at least 6.0%in the ethyl acetate extraction part of Crinum Asiaticum bulb.Part two:Study on the antiepileptic effect of the extract material from Crinum asiaticumObjective:To observe the antiepileptic effect of the extracts material from Crinum Asiaticum,the animal models were induced by electric stimulation and pentote-trazole compound.Methods:1 Maximal electroshock seizure test（MES）method:One hundred number mice（KM strain）,weight 18～22g,were divided into five groups randomly（n=20）and administrated by intra-gastric method of the following drugs:three groups with extracts dosage 17.2 mg/kg,33.4 mg/kg,66.8 mg/kg respectively,and one group with 25 mg/kg retigabine（RTG）,and control group with the solvent（0.5%CMC-Na）.The maximum electroshock protection rate was measured as the evaluation index.The multi-instrument（YLS-9A）was used to measure the antiepileptic ratio（also called protection ratio）,after administration 60 min and RTG 15 min.2 Metrazol maximal seizure model test（MMS）:One hundred number mice（KM strain）,were grouped by above the method,and administrated by intra-gastric once time,metrazol was applied to observe the threshold of generalized tonic-clonic seizures,and record the negative-respond number in30minutes by injecting PTZ s.c.85 mg/kg on the back or neck in mice.Results:1 Protection ratio of mice in MES test,RTG group 85%,three dosage extracts including low,middle and high groups respectively 25%,35%,60%,and had significant differences compare with the control group（P<0.05,P<0.01）.2 In MMS test,the high dose extracts and RTG groups compared with solvent group,had significant differences in the duration of the threshold induced generalized tonic-clonic seizures（P<0.05,P<0.01）.The protection ratio of extracts of Crinum Asiaticum were 5%,25%,30%,RTG was 50%,compared with solvent group themiddle and high dosage had significant difference（P<0.05）respectively.Summary:The extract of Crinum Asiaticum had a significant antiepileptic activity in the MES model,and also showed a certain antiepileptic effect in the MMS model mice administrated with middle and high dosage.Part three:The effects of the extracts of Crinum asiaticum and lylicine on KCNQ2 subtype potassium channelsObjective:To observe the effect of the extract of Crinum Asiaticum and the main ingredient of lycorine on KCNQ2 channel current.Methods:1 To construct a lentiviral vector plv-kcnq2 infected CHO cells,puromycin was used in screen the stable cell lines expressed KCNQ2 gene,and the Realtime PCR technique were used for detecting the target gene expression level.2 In CHO cell line expressed stable KCNQ2 gen,the effect of different concentration extract,1.5 mg/L,5 mg/L and 15 mg/L,on the channel current was observed by using the whole cell patch clamp method.3.Observe the effect of different concentrations of lycorine（μM/L）,0.1,1.0,3.0,10,30 and100,on the channel current of KCNQ2 with whole-cell patch clamp method.Results:1 The target KCNQ2 gene was over expressed 3.8x10⁷multiple times by realtime PCR detection.The electrophysiological parameters of channel were consistent with the reference.2 The extract of Crinum Asiaticum enhanced KCNQ2 channel current,and shifted left the half of the activation voltage(V_1/2).Using the concentration 15 mg/L extracts increased the amplitude of KCNQ2 current to1.3 times,and shifted the left V_1/2(△V_1/2)reached 14.2 mV maximally and accompanied by the change of channel dynamics.It shorten the activation parameter tau value(τ_act.)and prolonged the deactivation parameter tau value（τdeact.）.3 The lycorine had more potential to increase the KCNQ2 current,it enhanced the amplitude of currents to 1.55 times and EC₅₀0 was 5.24 uM.The lycorine shifted left the activation curve,the△V_1/2/2 value reached 26.4 mV,and also shorten the valueτ_act.,prolonged the valueτ_deacteact significantly.Summary:Extract of Crinum Asiaticum and the main alkaloid Lycorine activated the KCNQ2 channel currents concentration dependent and changed the channel kinetics parameters include shifting the activation curve to the left,accelerating the activation and slowing down the deactivation.Conclusions:The extract of Crinum Asiaticum showed an obvious antiepileptic activity,and contained 6%of the lycorine,which gained activation the KCNQ2 potassium channel.It turned out that the antiepileptic effect of the extract of Crinum Asiaticum could be related to containing the lycorine.