1、Anticonvulsant Effects of DrugsXXX（China Pharmaceutical University，639 Longmian Avenue, Nanjing, Jiangsu, China 210009）ABSTRACT: OBJECTIVE: To study the anticonvulsant efficacy of Phenytoin sodium and Phenobarbital and valproate on convulsion. METHODS: The anticonvulsant activity of the drugs was ev
2、aluated by the maximal electroshock (MES) test and remeflin seizure test. Induced rats were treated by intraperitoneal injection ,then observe the incubation period (up to tails upright 45) and degree of reaction of each mouse(spasm, falling down ,stiff or death). RESULTS: 1. In MES experiment, the
3、degree of convulsion: The Phenobarbital group (P0.05 vs normal saline group) was the lightest, and the NS group was the severest one. 2. In remeflin experiment, the similar result was observed between valproate group and NS group (P0.01). CONCLUSION: Phenobarbital has better anti-convulsion effect t
4、han Phenytoin sodium on convulsion induced by electroshock, and valproate has a significant effect to anti-convulsion induced by remeflin. KEYWORDS Phenytoin sodium; Phenobarbital; valproate; remeflin ; anti-convulsionIntroductionPhenytoin sodium: its mechanism of action: It can block sodium channel
5、s (voltage-, frequency-, and time dependent fashion) and inhibit the generation of action potentials and It can increase the function of inhibitory transmitter GABA, inhibit nerve terminal to uptake GABA and induce the increasing of GABA receptor, thereby enhance GABA-mediated postsynaptic inhibitio
6、n.Phenobarbital: its mechanism of action: Phenobarbital can inhibit the paradoxical discharge of epilepsy focus selectively, enhance stimulation of surrounding tissues and block discharge diffuse to normal tissues and Phenobarbital facilitate GABA-mediated inhibition of neuronal activity.Valproate i
7、s broad spectrum antiepileptic drug, which is used to all types of epilepsy; its mechanism is that enhancing the enzymatic activity of glutamate decarboxylase. And Inhibiting GABA reuptake and synapse inactivation via releasing synapse frontal membrane GABA and then enhance GABA postsynaptic inhibit
8、ionRemeflin, a central stimulate which can directly excite respiratory center, can produce convulsion with over-dose. The protective effects of drugs on convulsion produced by remeflin can be used to screen antiepileptic and anti-convulsion.Stimulating mouse head with a strong electric current can c
9、ause general tetanic convulsion. If a drug can prevent the occurrence of general titanic convulsion, we could initially suspect that it has antagonistic effect on grand mal. Remeflin, a central stimulant which can directly excite respiratory center, can protective effects of drugs on convulsion prod
10、uced by remeflin can be used to screen antiepileptics and anticonvulsants. 11 Material EquipmentsBalance, syringes, pharmacological and physiological multipurpose apparatusReagentsPhenytoin sodium, 0.5% solution w/v, Phenobarbital sodium, 0.5% solution w/v, 0.04% Remeflin, 2% sodium valporate, norma
11、l salineAnimals5mice, 18-22g, no restriction on sex2 Methods2.2 Methods of MES experiment Adjust the stimulator firstly. Stimulating parameters: working condition-electric shock; output voltage-maximum; model of stimulating-single; frequency-3216Hz; connect the outlet with clips and plug the connect
12、ion for power. Select convulsion mice. Attach the clips moistened with saline to the mouse ears. Punch the key to starting, and a current is applied to the head of the mouse, then cause tonic convulsion that is the state of the flexing forelimb and straightening hind limb. If the animals dont fall i
13、nto convulsion with 32Hz, modulate to 16Hz, and start again. Replace another one to test if have no reaction, yet. Select three mice in which convulsions occur typically. Mark and weigh them. Treat them by intraperitoneal injection of 5% phenytoin sodium 75mg/kg (0.15ml/10g), 5% phenobarbital sodium
14、 75mg/kg (0.15 ml/10g) and normal saline 0.15 ml/10g separately. 40 minutes after injection, stimulate the mice again with pro-threshold. Record the response.2.2 Methods of remeflin experimentMark and weigh two mice. Treat them separately by intraperitoneal injection of 2% sodium valproate 600mg/kg
15、(0.3ml/10g) and normal saline 0.3ml/10g. 30 minutes after administration, inject them with remeflin 8mg/kg (0.2ml/10g) subcutaneously. Observe the speed and degree of reaction of each mouse (spasm, falling down, stiff or death). 2.3 Statistic method and intensity measuresStatistic methods: Experimen
16、tal data in each groups were expressed by xs in the experiment used remeflin, analysis of variance and t-test (One-way Analysis of Variance, ANOVA) in the intergroup analysis , analysis of the intensity degree (Rank-Sum-Test ) results and analysis of rate by 2 test (Fishers exact test) were applied
17、statistical software SPSS 19.0, significant level =0.05.Outcome measures on behaviors in remeflin induced convulsion model: degree 0: no phenomenon; degree 1: tails upright; degree 2: tumble; degree 3: stiff; degree 4: death. 3 Results3.1 Results of MES experiment Phenobarbital was obvious to have t
18、he protective effects on MES convulsion (P0.05 vs Normal saline), and it is better than phenytoin sodium which has a weak anticonvulsant effiancy that the convulsion rate was between normal saline group and Phenobarbital group.Table 1 result of protective effects on MES convulsion of phenytoin sodiu
19、m and phenobarbital.Drugs Dosage（mg/kg） Convulsion/Sum Convulsion Rate(%)Normal saline 6/8 75Phenobarbital 75 1/8 12.5*Phenytoin sodium 75 4/8 50*P0.05 vs Normal saline3.2 Result of remeflin experimentThere was highly significant difference (P0.01) between the sodium valproate group and normal salin
20、e group in death rate, and significant difference (P0.05) between the two groups in convulsion incubation and reaction intensity. This means that sodium valproate has the anticonvulsant efficacy to play a protective effects on the convulsant induced by remeflin.After injection of remeflin, mice may
21、experienced incubation period, stiff flexion phase, straighting hind limb phase, clonic phase and restoration period. The mice in normal saline group behavior as follows: walked haltingly, jumped, uplifted fore limbs and stood on two feet, ears towards the back, and tails upwards like letter C. The
22、mice in sodium valproate group behavior as: Walked slowly and the tail was almost straight up. The whole body shaked, and after several minutes its hind limbs were stiff, and died.Table 2 the results of effects of sodium valproate on convulsion produced by remeflin (xs, n=8)Group Incubation (s) Deat
23、h Time (s) Death NumberDeath Rate (%)Normal Saline 4.181.18 No death 0 0Sodium Valproate 8.242.54* 7.680.80 # 6 75 *P0.01 vs Normal Saline group; * P0.01 vs Normal Saline group # except 2 mice that didnt dieTable3 the reaction intensity degree on mice convusion produced by remeflinGroup Reaction Int
24、ensity(Degree)Num 1 2 3 4 5 6 7 8Normal Saline 4 3 4 4 4 1 4 4Sodium Valproate* 1 1 2 1 1 1 1 1*P0.01 VS Normal Saline Group4 DiscussionsBecause of individual difference of animals, stimulating parameters should be set with experiment. Dont set it over-strong, in order to prevent animals from death.
25、 Prevent short circuit of clips carefully, otherwise the stimulator would be damaged. The convulsion of animals can be divided in five phase, latent period, stiff flexion phase, straightening hind limb phase, clonic phase and restoration period. If condition permits, pentetrazole is recommended to b
26、e substituted for remeflin because the convulsion caused by pententrazole is more typical.5 ConclusionsBoth Phenobarbital and phenytoin sodium have the protective effects on electric shock convulsion. And Phenobarbital whose effect is obvious has better anti-convulsion effect than phenytoin sodium w
27、hose effect is weak respectively on convulsion induced by electroshock. And valproate has a significant effect to anti-convulsion induced by remeflin, while remeflin can kill the most of the mice in experiment and valproate can reduce the toxicity of remeflin.Reference 1 Peng Xiaodong, et al Anticonvulsant efficacy of Chinese Materin Medicine Cynanchum Chinese R.Br in mice J J Fourth MilMed Univ 2009,30 (4):340-344
