Pharmacodynamics And Mechanism Of Action Study Of Anmeitang In Sleep-promoting

Insomnia is the perception or complaint of inadequate or poor-quality sleep because of one or more of the following:difficulty falling asleep, waking up frequently during the night with difficulty returning to sleep, waking up too early in the monring, sometimes even can not failing to sleep at all overnight. Insomnia often affects isomniacs's normal work, life, study and health. In the modem society, people are under a lot of pressures and stresses because their working rhythm is geting faster and faster. This is one reason why insomnia is so common. Meanwhile, insomnia makes people more stressful physically and psychologically. Because of the addiction, western medicine is restricted to be applied in the patients with chronic insomnia. Furthermore, Chinese traditional medicine(TCM) therapy of insomnia has obvious therapeutic effect and little or no side-effect, because it is based on the combination of disease and syndrome. Thus, recently, many researcher have focused on TCM study on insomnia.Anmtitang has been developed by professor ChenQun based on her decades clinic experiences and got good curative effect in clinic.In order to further to confirm the curative effect and the action mechanism, we designed this experimental stuty.Part 1 Literature summary and analysisThe article overviewed the differential treatment of ancient and modern in insomnia, the drug therapy of Chinese and Western medicines in treating insomnia and the progress of study in insomnia mechanism. Chinese traditional medicine has more than two thousand years of knowledge about insomnia. TCM doctors over past dynasties has stored up very plentiful knowledge and clinical experiences for this syndrome. They invented many effective therapeutics and formulas. They provided us very precis and plentiful literatures about insomnia.Through the literature summary and analysis,we think insomnia is typical disease caused by biological, psychological and social factor and that can induce multi-system and multi-hierarchy pathological rections. Thus mono-action and mono-target medication has some shortcomings inevitably and multi-hierarchy and multi-target traditional Chinese drug therapy may have some advantages. Because Chinese herb medicine compound has great advantage in multi-system, it is promising to study the application of Chinese herb medicine in the treatment of insomnia.Part 2 Experimental StudyExperiment I Anmeitang on the impact of the general behavior of miceObjective:To observed Anmeitang's effect on spontaneous activity and daily behavior of mice and to investigate the sedative effect of Anmeitang.Methods:The Anmeitang was given to mice intragastrically for 15 continuous days, and then the open country method was adopted to record the numbers of walking cell and lifting forelimb of mice within 2 minutes. Statistical process was performed on the recorded data. The differences between before and after administration in eating, drinking, defecation, coat color and independent activities were observed and compared among groups.Results:No significant difference was showed in eating, drinking, defecation, coat color and independent activities of each group before administration. After a period of administration, the mice of Anmeitang group showed significantly decreased in independent activities during the day, obviously increased in weight and were not easy to be disturbed in daytime sleeping. Compared with the control group, the Anmeitang low-dose group, the medium-dose group and the high-dose group, the diazepam control group and the Qiyeshenan tablets control group significantly reduced the numbers of activities in mice within 2 minutes (P<0.05).The Anmeitang medium-dose group significantly different from the low-dose group, the high-dose group and the Qiyeshen'an tablets control group (P<0.05), the Anmeitang medium-dose group was the most potent to reduce the numbers of activity of mice within 2 minutes. The gender differences in the numbers of activities within 2 minutes of mice were showed in the Anmeitang high-dose group (P<0.05), the numbers of activities within 2 minutes of female was more than that of male. The other groups have showed little gender differences within groups(P>0.05).Compared with the control group, Anmeitang low-dose group, Anmeitang medium-dose group and Anmeitang high-dose group and Diazepam control group significantly reduce the numbers of lifting forelimb of mice within 2 minutes (P< 0.05). The Anmeitang medium-dose group of is the most potent to reduce the numbers of lifting forelimb of mice within 2 minutes. Qiyeshenan tablets control group can reduce the numbers of lifting forelimb of mice within 2 minutes,but compared with the control group not significantly (P>0.05).ExperimentⅡSedative effect of AnmeitangObjective:Give pentobarbital sodium by intraperitoneal injection and observe the sedative-hypnotic effect of Anmeitang.Methods:The animals were randomly enrolled into 6 groups,12 mice in each group:control group, Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group, Diazepam control group, Qiyeshenan control group.The animal received the drug 20mg/kg, the animals in the control group received the same quantity of normal water once each day for 15 days. The mice were given pentobarbital 50mg/kg through intraperitoneal injection after the last drug administration. The time when righting reflex disappeared was recorded as drug-asleep time and the duration from drug-asleep to the recovery of righting reflex was recorded as sleep-time. Statistical process was performed on the recorded data.Results:The results indicate that Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group, Qiyeshenan control group and Diazepam control group can shorten mouse drop-asleep-time,compared with the control group significantly(P<0.05). Anmeitang medium-dose group can shorten mouse drop-asleep-time significantly, compared with Diazepam control group significantly(P<0.05). The gender differences on mouse drop-asleep-time were showed in the high-dose group of Anmeitang (P<0.05), the mouse drop-asleep-time of female is less than that of male. The other groups have showed little gender differences within groups(P>0.05).The results indicate that Anmeitang lower dose group, Anmeitang medium-dose group and Diazepam control group can prolong mouse sleep-time, compared with the control group significantly(P<0.05). Anmeitang high-dose group and Qiyeshenan control group can prolong mouse sleep-time, but compared with the control group not significantly(P>0.05).ExperimentⅢExploring the effect of Anmeitang on sleeping animals caused by pentobarbitalin subthreshold hypnosisdoseObjective:Give pentobarbital sodium subthreshold hypnosisdose by intraperitoneal injection and observe the sedative-hypnotic effect of Anmeitang.Methods:15 days after intragastric administration in mice, intraperitoneal injection of pentobarbital sodium subthreshold hypnosisdose and then record the number of sleeping mice.Results:The number of sleeping mice in normal control group is 1 and incidence rate is 8.3%; the number of sleeping mice in Anmeitang low-dose group is 2 and incidence rate is 16.7%; in Anmeitang medium-dose group is 8 and incidence rate is 66.7%; in Anmeitang high-dose group is 6 and incidence rate is 50%; in Diazepam control group is 4 and incidence rate is 33.3%; in Qiyeshenanpian control group is 4 and incidence rate is 33.3%. Compared with normal control group, Anmeitang medium-dose group, high-dose group, Diazepam control group and Qiyeshenanpian control group can increase the quantity of sleeping animals caused by pentobarbitalin sodium subthreshold hypnosisdose and improve the incidence rate of sleeping mice significantly (P<0.05).ExperimentⅣExploring the effect of Anmeitang against strychnine-induced convulsion in miceObjective:Investigate the anticonvulsant effect of Anmeitang in mice.Methods:15 days after intragastric administration in mice, intraperitoneal injection of strychnine nitrate and then record the number of eclamptic mice.Results:The number of eclamptic mice in normal control group is 12 and incidence rate is 100%; the number of eclamptic mice in Anmeitang low-dose group is 8 and incidence rate is 66.7%; in Anmeitang medium-dose group is 3 and incidence rate is 25.0%; in Anmeitang high-dose group is 2 and incidence rate is 16.7%; in Diazepam control group is 1 and incidence rate is 8.3%; in Qiyeshenanpian control group is 8 and incidence rate is 66.7%. Anmeitang low-dose group, medium-dose group, high-dose group, Diazepam control group and Qiyeshenanpian control group compared with normal control group respectively, P<0.05, which shows statistical significance as well as the different incidence rate of convulsion in mice, that is the incidence rate of convulsion in the former five groups is obviously lower than normal control group.Experiment V Anmeitang on the impact of the central neurotransmitter of insomnia miceObjective:To detect the content of 5-HT,5-HIAA, NE, DA in insomnia ra(?)s brain and to investigate the mechanism of Anmeitang on improving the insomnia.Methods:A high performance liquid chromatography with electro-chemical (HPLC-EC) detection method was adopted to detect the content of 5-HT,5-HIAA, NE, DA in insomnia rats brain. The experimental model of insomnia rats was induced by PCPA..Chromatographic conditions:Column:Phenomenex column (Luna C18,150×4.6mm),5μm, Alltech precolumn (Alltima C185μ, Guard 7.5×4.6mm)Mobile phase:acetonitrile:water phase (13:87), the water phase was adjusted to PH 4.0 with phosphoric acid.Flow rate:1.0ml/min.Set Voltage:+350 mv.Column temperature:35℃.Results:The blank model control group and blank control group showed a difference (P<0.05), the content of 5-HT in rats brain of the blank model control group was significantly reduced. Compared with the blank model control group, the Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group, and the diazepam control group improved the content of 5-HT in insomnia rats brain, while the Anmeitang high-dose group and the diazepam control group significantly improved the content of 5-HT in insomnia rats brain (P<0.05). Comparing with the content of 5-HT between male and female in each group showed little difference in gender. (P>0.05)The blank model control group and blank control group showed a difference (P <0.05), the content of 5-HIAA in rats brain of the blank model control group was significantly reduced. Compared with the blank model control group, the Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group, and the diazepam control group can improve the content of 5-HIAA in insomnia rats brain, while the Anmeitang high-dose group can improve the content of 5-HIAA in insomnia rats brain significantly (P<0.05). The Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group showed little difference between groups. Comparing with the content of 5-HIAA between male and female in each group showed little difference in gender. (P>0.05)Compared with the blank control group, the content of NE in rats brain of the blank model control group was increased, but the difference were not significantly(P>0.05). Compared with the blank model control group, the Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group, and the diazepam control group can decrease the content of NE in insomnia rats brain, but the difference were not significantly(P>0.05). The Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group showed little difference between groups. The gender differences in the content of NE in insomnia rats brain were showed in the Anmeitang high-dose group (P<0.05), the content of NE in insomnia rats brain of female was more than that of male. The other groups have showed little gender differences within groups(P>0.05).The blank model control group and blank control group showed a difference (P <0.05), the content of DA in rats brain of the blank model control group was significantly increased. Compared with the blank model control group, the Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group, and the diazepam control group can decrease the content of DA in insomnia rats brain, while the Anmeitang medium-dose group and Anmeitang high-dose group can decrease the content of DA in insomnia rats brain significantly (P<0.05). The Anmeitang low-dose group, Anmeitang medium-dose group, Anmeitang high-dose group showed little difference between groups. Comparing with the content of DA between male and female in each group showed little difference in gender. (P>0.05)Part 3 Conclusion:1. Anmeitang has notable effect on the independent activities of mice. It can remarkably reduce independent activities in mice, and that shows its obvious sedative effect which is better than Diazepam and Qiyeshenanpian.2. Anmeitang has notable effect on sleep latency and sleep time of mice. It can remarkably curtail sleep latency and prolong sleep time, and that shows its obvious hypnotic effect which is better than Diazepam and Qiyeshenanpian.3. Anmeitang's effect on improving slumber incidence rate in mice is superior to Diazepam control group and Qiyeshenanpian control group, and its effect on reducing incidence rate of convulsion in mice is superior to Qiyeshenanpian control group but is inferior to Diazepam control group.4. The comparative result of Anmeitang of high, medium and low doses shows that Anmeitang medium dose group has the most obvious effect on independent activities in mice, Anmeitang low and medium dose groups have more obvious effect on sleep latency and sleep time in mice, Anmeitang medium and high dose groups have more obvious effect on slumber incidence rate in mice as well as on reducing incidence rate of convulsion in mice, and medium dose can be used as reference dose of drug development and clinical application.5. The sedative effect and hypnotic effect of Anmeitang is not obviously different in mice of different gender, which point out that the effect of Anmeitang has no gender differences.6. Through study of the effect of intracephalic neurotransmitter content in insomnious mice, we find that Anmeitang can notably increase 5-HT content in insomnious mice's brain, and reduce DA content. Anmeitang might act on slumber-awakening system by increasing 5-HT content and reducing DA content, so as to increase sleep time and improve sleep quality.7. This experiment adopts HPLC-EC method to establish a fast, accurate and stable detection method of monoamine neurotransmitter content detection. The result shows that test index components all tested in 10 minutes. The linear relation between each peak area and neurotransmitter consistency is good, the precision of each test group is good, the solvent peak has nil interference to each sample test component under appointed chromatographic condition, and four test components of each sample solution can all achieve baseline separation.