| Objective:To study conserving effect and reducing toxicity integrated mechanism of the effect/toxicity amphiprotic components from Ma-Xing-Shi-Gan Prescription (MXP) on central nervous system.Methods:(1) Acute toxicity experiment:Mice were intragastric (ig) administrated with different dose Ma Huang (MH), Honey-fried Ma Huang (HMH) and MXP. The mice were observed for 7d and death rates were recorded. LD50 and confidence interval of 95% LD50 of above mentioned drugs were calculated with Bliss software.(2) Autonomic activities experiment:Mice(♂) were divided into 11 groups, namely normal saline group(NS),ephedrine group(EP), high dose Ma Huang group(MH-H), moderate dose Ma Huang group(MH-M), low dose Ma Huang group(MH-L), high dose Honey-fried Ma Huang group(HMH-H), moderate dose Honey-fried Ma Huang group(HMH-M), low dose Honey-fried Ma Huang group(HMH-L), high dose MXP group (MXP-H), moderate dose MXP group (MXP-M) and low dose MXP group (MX-L). The numbers of autonomic activitiy in 15 minutes before ig, and after ig 30minutes, 1h,2h,3h,4h were determinated. The results were handled with SPSS13.0 software.(3) Pentobabital sodium sleep experiment:Male Kunming mice were divided into 11 groups, namely NS, EP,3 dose (high, medium, and low) MH,3 dose HMH, and 3 dose MXP. The corresponding drugs were administered intragastrically for 6 consecutive days, and 45 min after the final administration, the mice received intraperitoneal injection (ip) of pentobabital sodium, and the latent period and continuous sleeping time were recorded. The results were handled with one-way ANOVA from SPSS 13.0 software.(4)Open field experiment:Male Kunming mice were divided into 11 groups, namely NS, EP,3 dose (high, medium, and low) MH,3 dose HMH, and 3 dose MXP. The corresponding drugs were administered intragastrically for 7 consecutive days. 45 min after the final administration, the mice were set in the center of a open wooden case, and spanning grid frequency, standing frequency and decorating frequency were recorded. The results were handled with one-way ANOVA from SPSS 13.0 software.(5) HE staining,5-hydroxytryptamine(5-HT) and norepinephrine(NE) immunohistochemistry experiments:HE staining:Male SD rats were divided into 11 groups, namely NS, EP,3 dose (high, medium, and low) relatively fresh MH(FMH), 3 dose stale MH(SMH), and 3 dose MXP. The corresponding drugs were administered intragastrically for 6 consecutive days.1h after the final administration, brains were obtained by heart perfusion, and fixed with 10% neutrality formaldehyde, routinely dehydrated, cleared, embedded with paraffin wax, coronally sliced. The sections comprising frontal cortex and hippocampus were stainned by HE,5-HT and NE immunohistochemistry. The effects of FMH, SMH and MXP on brain tissue structure, and the expression of 5-HT and NE in frontal cortex and hippocampus were studied.(6) The effects of FMH, SMH and MXP on the contents of four amino acid neurotransmitters in different brain regions:Group and administration were same as (5). 1h after the final ig, brains were obtained, and frontal cortex, hippocampus, hypothalamus and brain stem were quickly separated on ice-box. The contents of glutamic acid (Glu), aspartic acid(Asp), gamma aminobutyric acid(GABA) and glycine(Gly) were determined by LC-ESI+-MS-MS. The results were handled with one-way ANOVA from SPSS 13.0 software.(7) The comparison of the contents of ephedrine category alkaloid in FMH, SMH and MXP after ig:Group and administration were same as (5). 1h after the final ig., brains were obtained, and frontal cortex, hippocampus, hypothalamus and brain stem were quickly separated on ice-box. The contents of ephedrine (EP), pseudoephedrine (PEP), norephedrine (NEP) and norpseudoephedrine (NPEP) were determined by LC-ESI+-MS-MS. The results were handled with one-way ANOVA from SPSS 13.0 software.Results:(1) Acute toxicity experiment:LD50 of MH, HMH and MXP were 134.88g/kg,204.87g/kg,77.42g/kg, respectively.95% confidence interval were 118.56-156.99g/kg,174.46-236.28g/kg,63.009-123.82g/kg, respectively. In the experiment, MH were administered for one time, and HMH and MXP were administered for two times (interval time:4h).(2) Autonomic activities experiment:Main effect and interactive effect analysis:Noticeable differences existed among different time (F=99.289, P=0.000). Interactive effect existed between time and group (F= 1.497,P=0.043). Noticeable differences exist among different group (F=2.742,P=0.005). The results of LSD multiple comparison of different levels of time factor showed that noticeable differences existed between before ig and other 5 time (all P=0.000).Solitude effect analysis of group factor:30min after ig, compared with MH-L, autonomic activity of HMH-L were reduced, and the difference had no signifance, P=0.989, so did that of MXP-L, P=0.860. lh after ig, compared with MH-L, autonomic activity of HMH-L were reduced, and the difference had no signifance, P=0.867.2h after ig, compared with MH-L, autonomic activity of HMH-L were reduced, and the difference had no signifance, P=0.996.4h after ig, compared with MH-L, autonomic activity of MXP-L were reduced, and the difference had no signifance,P=1.000.Solitude effect analysis of time factor:Autonomic activity of MXP-H among different time had no significant difference, P=0.119. Compared with before ig, autonomic activities of MH-L in other 5 time were reduced, the difference was significant, P was equal to 0.005,0.003,0.001,0.001,0.000, relatively. Compared with 30min after ig, autonomic activity of MH-L 3h after ig were reduced, the difference was significant, P=0.011. Compared with ig ih later, autonomic activity of MH-L at ig 3h later were reduced, the difference was significant, P=0.002. Compared with before ig, autonomic activities of HMH-L in other 5 time were reduced, the difference was significant, P was equal to 0.001,0.001,0.000,0.000,0.007. Compared with 30min after ig, autonomic activity of HMH-L at ig 2h later were reduced, the difference was significant, P=0.008.(3) Pentobabital sodium sleep experiment:Compared with MH-H, MXP-H increased sleep time, the difference was significant, P=0.018, and HMH-H increased sleep time, the difference had no significance, P=0.060. Compared with MH-M, MXP-M increased sleep time, the difference was significant,P=0.029, and HMH-M increased sleep time, the difference had no significance, P=0.259. Compared with MH-L, MXP-L increased sleep time, the difference was significant, P=0.000, and HMH-L increased sleep time, the difference had no significance, P=0.592. Compared with NS, MXP-M reduced sleep time, the difference had no significance, P=0.355, and MXP-L incresed sleep time, the difference had no significance, P=0.592. Compared with NS, all of EP,3 dose MH,3 dose HMH and MXP-H reduced sleep time, the difference was significant, P was equal to 0.036,0.000,0.002,0.001,0.008, 0.000,0.000,0.029. The whole comparison of the sleep latency had no significant difference, F=0.841, P=0.593.(4)Open field experiment:Compared with MH-H, MXP-H increased spanning grid, the difference was significant,P=0.000. The whole comparison of standing frequency had no significant difference, F=1.306, P=0.238. The whole comparison of decorating frequency had no significant difference, F=1.667,P=0.124.(5)HE staining,5-HT and NE immunohistochemistry experiments:HE staining: Obvious neuron apoptosis was visible in frontal cortex and hippocampus of 3 dose FMH. Compared with corresponding dose FMH,3 dose SMH and 3 dose MXP obviously reduded apoptosis neuron.5-HT immunohistochemistry:5-HT positive neurons were difficultly visible in frontal cortex and hippocampus of 3 dose FMH. Compared with corresponding dose FMH, SMH-H and SMH-M increased amount of 5-HT positive neuron, deepened color of 5-HT positive neuron in frontal cortex and hippocampus; so did SMH-L, MXP-H, MXP-L, but only in frontal cortex. NE immunohistochemistry:Compared with NS,3 dose FMH increased amount of NE positive neuron, deepened color of NE positive neuron only in frontal cortex. Compared with corresponding dose FMH,3 dose SMH and 3 dose MXP reduced amount of NE positive neuron only in frontal cortex, and color of the NE positive neuron getted faint.(6) The effects of FMH, SMH and MXP on the content of four amino acid neurotransmitters in different brain regions:Compared with FMH-H, SMH-H reduced the content of Glu, Asp and Gly in frontal cortex, the difference was significant, all of P were equal to 0.000. Compared with FMH-M, both of SMH-M and MXP-M reduced the content of Glu in frontal cortex, the differences were significant, P were equal to 0.010,0.015, respectively. Compared with FMH-L, both of SMH-L and MXP-L reduced the content of Glu in frontal cortex, the differences were significant, both of P were equal to 0.000. Compared with SMH-H, MXP-H increased the content of GABA in frontal cortex, the differences were significant, P=0.036.Compared with FMH-H, SMH-H reduced the contents of Glu, GABA and Gly in hippocampus, the differences were significant, P were equal to 0.017,0.000,0.000, respectively; MXP-H reduced the contents of GABA and Gly in hippocampus, the differences were significant, P were equal to 0.005,0.027, respectively. Compared with FMH-M, SMH-M reduced the contents of Glu and GABA in hippocampus, the differences were significant, P were equal to 0.001,0.012, respectively. MXP-M reduced the contents of Glu and GABA in hippocampus, the differences were significant, P were equal to 0.012,0.038, respectively. Compared with FMH-L, SMH-L reduced the contents of Glu, GABA and Gly in hippocampus, the differences were significant, P were equal to 0.037,0.000,0.002, respectively; MXP-L reduced the contents of GABA and Gly in hippocampus, the differences were significant, P were equal to 0.000,0.011, respectively. Compared with SMH-H, MXP-H increased the content of Gly in hippocampus, the difference was significant,P=0.006. Compared with FMH-H, both of SMH-H and MXP-H increased Glu/GABA, the differences were significant, P were equal to 0.010,0.017, respectively. Compared with FMH-M, both of SMH-M and MXP-M increased Glu/GABA, the difference had no significance, P were equal to 0.579,0.619, respectively. Compared with FMH-L, both of SMH-L and MXP-L increased Glu/GABA, the differences were significant, P were equal to 0.001,0.000, respectively.Compared with FMH-H, SMH-H reduced the contents of Glu, Asp, GABA and Gly in hypothalamus, the differences were significant, P were equal to 0.003,0.003, 0.008,0.014, respectively; MXP-H reduced the contents of Glu, Asp, GABA and Gly in hypothalamus, the differences were significant, P were equal to 0.001,0.006,0.010, 0.004, respectively. Compared with FMH-M, SMH-M reduced the contents of Glu and Gly in hypothalamus, the differences were significant, P were equal to 0.000, 0.029, respectively; MXP-M reduced the contents of Glu and Gly in hypothalamus, the differences were significant, P were equal to 0.007,0.022, respectively. Compared with FMH-L, SMH-L reduced the content of Glu in hypothalamus, the difference was significant,.P=0.003; MXP-L reduced the contents of Glu and Gly in hypothalamus, the differences were significant, P were equal to 0.001,0.012, respectively. Compared with FMH-H, both of SMH-H and MXP-H increased Glu/GABA in hypothalamus, the differences had no significance, P were equal to 0.0794,1.000, respectively. Compared with FMH-M, both of SMH-M and MXP-M decreased Glu/GABA in hypothalamus, the differences had no significance, P were equal to 0.781,0.671, respectively. Compared with FMH-L, both of SMH-L and MXP-L decreased Glu/GABA in hypothalamus, the differences had no significance, P were equal to 0.690,1.000.The whole comparison of the content of Glu in brain stem had no significance, F=0.625, P=0.790. So did the content of Asp, GABA, Gly and Glu/GABA in brain stem, F=0.504, P=0.0.884 (Asp), F=1.345, P=0.241(GABA), F=1.169, P=0.321 (Gly), F=1.289, P=0.247 (Glu/GABA).(7) The comparison of the contents of ephedrine category alkaloid in FMH, SMH and MXP after ig:Compared with corresponding dose FMH, the contents of EP, PEP, NEP and NPEP in frontal cortex from 3 dose SMH and 3 dose MXP had no significant change.Conclusions:(1) According to acute toxicity, HMH |
PDF Full Text Request