安寐汤促睡眠药效研究及机理初探
摘要
失眠,中医又称为“不寐”、“不得眠”、“不得卧”、“目不瞑”等,是以经常不能获得正常睡眠为特征的一类病症,主要表现为睡眠时间、深度的不足,轻者入寐困难,或寐而易醒,或醒后不能再寐,重则彻夜不寐,常影响人们正常的工作、生活、学习和健康。目前西药常用催眠剂与安定剂,可产生即时疗效,但药物的毒副作用明显,且长期服药会使病人对药物的耐受性增加,并产生对药物的依赖性。祖国医学在失眠治疗上积累了丰富的经验,中药以其副作用小也显示出明显的优势。因此,加强中医药的开发利用,研究出具有自主知识产权的有效中药制剂是当务之急。
多年来,导师陈群教授在中医理论指导下,总结前人宝贵经验,结合失眠病因病机,在长期的临床实践中拟定出治疗失眠的基础方剂-安寐汤,取得良好的临床疗效。安寐汤组成为：夜交藤、酸枣仁、生龙骨、珍珠母、五味子、白芍、生地。安寐汤方中夜交藤药性平和,功擅引阳入阴；生龙骨、珍珠母含钙、铁、钠、钾等微量元素,可抑制神经与骨骼肌兴奋,作用于REM时期,对失眠伴有多梦、惊悸、遗精、盗汗效好。五味子含五味子素、五仁醇,与白芍均对中枢神经系统有抑制、镇静作用。酸枣仁对实验动物和人均有显著而温和的催眠、镇静、安定效果,为治疗虚烦不得眠的要药。酸枣仁与夜交藤两药,为无论古代医家还是现代中医治疗失眠使用最多的药物,也是安寐汤的君药。
通过对该方的分析可知,安寐汤药味精炼、组方巧妙合理,治标当中兼顾治本,本方可治疗各种失眠,具有宁神安寐、诱导睡眠的作用。为进一步探讨安寐汤的疗效和作用机理,我们进行了安寐汤的实验研究,研究结果可为安寐汤的进一步开发研究提供依据,为今后研制出疗效确切、使用简便的中药制剂奠定基础。
第一部分文献综述与分析
文献综述了失眠的古今论治、中西医药物治疗失眠的概况及失眠机理研究进展等三大方面的内容。祖国医学对不寐病证的认识己有两千多年的历史,历代医家积累了极其丰富的认识和治疗经验,创立了不少行之有效的治法和方药,为我们治疗失眠留下了极为宝贵和丰富的文献资料。通过对文献综述分析的结果,我们认为失眠是一种典型的生物、心理、社会等多种因素作用而致的疾病,可引起人体多系统、多层次的病理反应,因而单一作用、靶点明确的药物治疗不可避免地具有一些缺点,而多层次、多靶点的中医中药疗法更具优势。
第二部分实验研究
(一)安寐汤对小鼠一般行为学的影响
研究目的：观察安寐汤对小鼠自主活动及日常行为的影响,探讨安寐汤的镇静作用效果。
研究方法：小鼠灌胃给药15天后,采用旷野法记数2分钟内的行走格数和前肢向上抬举次数,结果进行统计分析。观察比较各组小鼠给药前后进食、饮水、排便、毛色及自主活动等一般情况的差异。
研究结果：给药前各组小鼠的进食、饮水、排便、毛色及自主活动等方面均无明显差异。给药一段时间后,安寐汤给药组小鼠白天自主活动明显减少,体重增加较为明显,在白天睡眠时不容易被打扰。
与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组和七叶神安片对照组能显著降低小鼠2分钟内活动次数(P<0.05)安寐汤中剂量组分别与安寐汤低剂量组、安寐汤高剂量组、七叶神安片对照组比较差异明显(P<0.05),降低小鼠2分钟内活动次数作用最强。安寐汤高剂量组对老鼠2分钟内活动次数的影响存在性别差异(P<0.05),雌老鼠2分钟内活动次数要高于雄老鼠。其余各组尚不能认为存在组内性别差异(P>0.05)。
与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组能显著降低小鼠2分钟内上举前肢的次数(P<0.05)。安寐汤中剂量组分别与安寐汤低剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组比较差异明显(P<0.05),对降低小鼠2分钟内上举前肢的次数作用最强。七叶神安片对照组对降低小鼠2分钟内上举前肢次数作用不显著(P>0.05)。
(二)安寐汤对阈上剂量戊巴比妥钠致小鼠睡眠时间的影响
研究目的：观察安寐汤对阈上剂量戊巴比妥钠的协同作用,探讨安寐汤促睡眠药效作用。
研究方法：小鼠灌胃给药15天后,腹腔注射阈上剂量戊巴比妥钠,记录各组入睡潜伏期和睡眠维持时间,结果进行统计学处理。
研究结果：与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组和七叶神安片对照组能显著降低小鼠入睡潜伏期(P<0.05)。安寐汤中剂量组与安定对照组差异明显(P<0.05),安寐汤中剂量组对降低小鼠入睡潜伏期作用更强。安定对照组对老鼠入睡潜伏时间的影响存在性别差异(P<0.05),雌老鼠入睡潜伏期短于雄老鼠。其余各组尚不能认为存在组内性别差异。
与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安定对照组能显著延长小鼠睡眠维持时间(P<0.05)。安寐汤高剂量组对小鼠睡眠维持时间作用不显著(P>0.05)。七叶神安片对照组对小鼠睡眠维持时间作用不显著(P>0.05)
(三)安寐汤对阈下剂量戊巴比妥钠致小鼠睡眠的影响
研究目的：观察安寐汤对阈下剂量戊巴比妥钠的协同作用,探讨安寐汤促睡眠药效作用。
研究方法：小鼠灌胃给药15天后,腹腔注射阈下剂量戊巴比妥钠,记录各给药组入睡率,结果进行统计学处理。
研究结果：正常对照组睡眠小鼠的数量为1只,发生率为8.3%；安寐汤低剂量组睡眠小鼠的数量为2只,发生率为16.7%；安寐汤中剂量组为8只,66.7%；安寐汤高剂量组为6只,50%；安定对照组为4只,33.33%；七叶神安片对照组为4只,33.33%。安寐汤中剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组分别与正常对照组比较有统计学意义(P<0.05),安寐汤中剂量组,安寐汤高剂量组,安定对照组,七叶神安片对照组小鼠睡眠发生率明显高于正常对照组。
(四)安寐汤对抗士的宁致小鼠惊厥的作用研究
研究目的：探讨安寐汤对小鼠的抗惊厥作用。
研究方法：小鼠灌胃给药15天后,腹腔注射硝酸士的宁,记录小鼠惊厥数。结果进行统计学分析。
研究结果：正常对照组发生惊厥小鼠的数量为12只,发生率为100%；安寐汤低剂量组睡眠小鼠的数量为8只,发生率为66.7%；安寐汤中剂量组为3只,25.0%；安寐汤高剂量组为2只,16.7%；安定对照组为1只,8.3%；七叶神安片对照组为8只,66.7%。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组分别与正常对照组比较有统计学意义(P<0.05),安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组小鼠惊厥发生率明显低于正常对照组。
(五)安寐汤对失眠大鼠脑内中枢神经递质的影响
研究目的：检测安寐汤对失眠大鼠脑内5-HT、5-HIAA、NE、DA的含量的影响,探讨安寐汤改善失眠作用机制。
研究方法：实验用PCPA造成失眠大鼠模型,采用高效液相-电化学检测法检测失眠大鼠脑内5-HT、5-HIAA、NE、DA的含量变化。色谱条件为：色谱柱采用Phenomenex色谱柱(Luna C18,150×4.6mm,5μm); Alltech预柱(Alltima C185μm,Guard 7.5×4.6mm)。流动相采用乙腈：水相=13：87,用磷酸调水相的pH为4.0。流速为1.0ml／min。设定电压为+350mv。柱温为35℃。
研究结果：空白对照组与空白模型对照组比较有差异(P<0.05),空白模型对照组大鼠脑内5-HT含量显著降低。与空白模型对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组均能提高失眠大鼠脑内5-HT含量,其中安寐汤高剂量组、安定对照组能显著提高失眠大鼠脑内5-HT含量(P<0.05)。各组雌雄老鼠脑内5-HT含量的疗效比较,P>0.05,尚不能认为存在组内性别差异。
与空白对照组相比,空白模型对照组大鼠脑内5-HIAA含量显著降低(P<0.05)。与空白模型对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组均能提高失眠大鼠脑内5-HIAA含量,其中安寐汤高剂量组能显著提高失眠大鼠脑内5-HIAA含量(P<0.05)。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组间差异不明显。各组雌雄老鼠脑内5-HIAA含量的疗效比较,P>0.05,尚不能认为存在组内性别差异。
与空白对照组相比,空白模型对照组大鼠脑内NE含量有所升高。空白模型组大鼠脑内NE含量是较空白组有所增加,但两组相比无显著性差异。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组可降低空白模型对照组大鼠脑内升高的NE含量,但作用不显著。安寐汤高剂量组对失眠大鼠脑内NE含量的影响存在性别差异(P<0.05),雌老鼠脑内NE的含量低于雄老鼠。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组间差异不明显。
与空白对照组相比,空白模型对照组大鼠脑内DA含量显著升高(P<0.05)。与空白模型对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组均能降低失眠大鼠脑内DA含量,其中安寐汤中剂量组、安寐汤高剂量组能显著降低失眠大鼠脑内DA含量(P<0.05)。
第三部分研究结论
1.安寐汤对小鼠自主活动影响效果显著,可显著降低小鼠的自主活动性,说明安寐汤镇静效果明显,镇静作用优于安定和七叶神安片。
2.安寐汤对小鼠入睡潜伏期和睡眠时间影响效果显著,可显著缩短入睡潜伏期和延长睡眠时间,说明安寐汤催眠效果明显,催眠作用优于安定和七叶神安片。
3.安寐汤在提高小鼠睡眠发生率的作用上优于安定对照组和七叶神安片对照组,在降低小鼠惊厥发生率的作用上优于七叶神安片对照组,但比安定对照组作用效果弱。
4.安寐汤高、中、低不同剂量组之间的疗效对比结果表明,安寐汤各剂量组对小鼠自主活动影响效果以安寐汤中剂量组效果最为明显,对小鼠入睡潜伏期和睡眠时间影响效果以安寐汤低、中剂量组效果较为明显,对小鼠睡眠发生率作用以安寐汤中、高剂量组效果较为明显,对降低小鼠惊厥发生率作用以安寐汤中、高剂量组效果较为明显,提示中剂量可作为药物开发及临床用药的参考剂量。
5.安寐汤对不同性别老鼠镇静作用、催眠作用差异均不明显,提示安寐汤作用无性别上的差异。
6.通过对失眠大鼠脑内中枢神经递质含量的作用进行研究,我们发现安寐汤可以显著增加失眠大鼠脑内五羟色胺、五羟吲哚乙酸的含量,降低多巴胺的含量。安寐汤可能是通过增加五羟色胺、五羟吲哚乙酸的含量,降低多巴胺的含量而作用于睡眠—觉醒机制,以实现增加睡眠、改善睡眠的作用。
7.本实验采用高效液相-电化学检测法建立了快捷、准确、稳定的检测单胺类中枢神经递质含量的检测方法。结果表明,待测指标成分在10分钟内全部检测完成。各峰面积与神经递质浓度线性关系良好,各待测组分日内精密度良好,拟定的色谱条件下溶剂峰对样品各待测成分无干扰,各样品溶液中四个待测成分均能达到基线分离。
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 analysis
The 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 Study
Experiment I Anmeitang on the impact of the general behavior of mice
Objective: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 Anmeitang
Objective: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 hypnosisdose
Objective: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 mice
Objective: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 mice
Objective: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.
多年来,导师陈群教授在中医理论指导下,总结前人宝贵经验,结合失眠病因病机,在长期的临床实践中拟定出治疗失眠的基础方剂-安寐汤,取得良好的临床疗效。安寐汤组成为：夜交藤、酸枣仁、生龙骨、珍珠母、五味子、白芍、生地。安寐汤方中夜交藤药性平和,功擅引阳入阴；生龙骨、珍珠母含钙、铁、钠、钾等微量元素,可抑制神经与骨骼肌兴奋,作用于REM时期,对失眠伴有多梦、惊悸、遗精、盗汗效好。五味子含五味子素、五仁醇,与白芍均对中枢神经系统有抑制、镇静作用。酸枣仁对实验动物和人均有显著而温和的催眠、镇静、安定效果,为治疗虚烦不得眠的要药。酸枣仁与夜交藤两药,为无论古代医家还是现代中医治疗失眠使用最多的药物,也是安寐汤的君药。
通过对该方的分析可知,安寐汤药味精炼、组方巧妙合理,治标当中兼顾治本,本方可治疗各种失眠,具有宁神安寐、诱导睡眠的作用。为进一步探讨安寐汤的疗效和作用机理,我们进行了安寐汤的实验研究,研究结果可为安寐汤的进一步开发研究提供依据,为今后研制出疗效确切、使用简便的中药制剂奠定基础。
第一部分文献综述与分析
文献综述了失眠的古今论治、中西医药物治疗失眠的概况及失眠机理研究进展等三大方面的内容。祖国医学对不寐病证的认识己有两千多年的历史,历代医家积累了极其丰富的认识和治疗经验,创立了不少行之有效的治法和方药,为我们治疗失眠留下了极为宝贵和丰富的文献资料。通过对文献综述分析的结果,我们认为失眠是一种典型的生物、心理、社会等多种因素作用而致的疾病,可引起人体多系统、多层次的病理反应,因而单一作用、靶点明确的药物治疗不可避免地具有一些缺点,而多层次、多靶点的中医中药疗法更具优势。
第二部分实验研究
(一)安寐汤对小鼠一般行为学的影响
研究目的：观察安寐汤对小鼠自主活动及日常行为的影响,探讨安寐汤的镇静作用效果。
研究方法：小鼠灌胃给药15天后,采用旷野法记数2分钟内的行走格数和前肢向上抬举次数,结果进行统计分析。观察比较各组小鼠给药前后进食、饮水、排便、毛色及自主活动等一般情况的差异。
研究结果：给药前各组小鼠的进食、饮水、排便、毛色及自主活动等方面均无明显差异。给药一段时间后,安寐汤给药组小鼠白天自主活动明显减少,体重增加较为明显,在白天睡眠时不容易被打扰。
与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组和七叶神安片对照组能显著降低小鼠2分钟内活动次数(P<0.05)安寐汤中剂量组分别与安寐汤低剂量组、安寐汤高剂量组、七叶神安片对照组比较差异明显(P<0.05),降低小鼠2分钟内活动次数作用最强。安寐汤高剂量组对老鼠2分钟内活动次数的影响存在性别差异(P<0.05),雌老鼠2分钟内活动次数要高于雄老鼠。其余各组尚不能认为存在组内性别差异(P>0.05)。
与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组能显著降低小鼠2分钟内上举前肢的次数(P<0.05)。安寐汤中剂量组分别与安寐汤低剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组比较差异明显(P<0.05),对降低小鼠2分钟内上举前肢的次数作用最强。七叶神安片对照组对降低小鼠2分钟内上举前肢次数作用不显著(P>0.05)。
(二)安寐汤对阈上剂量戊巴比妥钠致小鼠睡眠时间的影响
研究目的：观察安寐汤对阈上剂量戊巴比妥钠的协同作用,探讨安寐汤促睡眠药效作用。
研究方法：小鼠灌胃给药15天后,腹腔注射阈上剂量戊巴比妥钠,记录各组入睡潜伏期和睡眠维持时间,结果进行统计学处理。
研究结果：与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组和七叶神安片对照组能显著降低小鼠入睡潜伏期(P<0.05)。安寐汤中剂量组与安定对照组差异明显(P<0.05),安寐汤中剂量组对降低小鼠入睡潜伏期作用更强。安定对照组对老鼠入睡潜伏时间的影响存在性别差异(P<0.05),雌老鼠入睡潜伏期短于雄老鼠。其余各组尚不能认为存在组内性别差异。
与正常对照组相比,安寐汤低剂量组、安寐汤中剂量组、安定对照组能显著延长小鼠睡眠维持时间(P<0.05)。安寐汤高剂量组对小鼠睡眠维持时间作用不显著(P>0.05)。七叶神安片对照组对小鼠睡眠维持时间作用不显著(P>0.05)
(三)安寐汤对阈下剂量戊巴比妥钠致小鼠睡眠的影响
研究目的：观察安寐汤对阈下剂量戊巴比妥钠的协同作用,探讨安寐汤促睡眠药效作用。
研究方法：小鼠灌胃给药15天后,腹腔注射阈下剂量戊巴比妥钠,记录各给药组入睡率,结果进行统计学处理。
研究结果：正常对照组睡眠小鼠的数量为1只,发生率为8.3%；安寐汤低剂量组睡眠小鼠的数量为2只,发生率为16.7%；安寐汤中剂量组为8只,66.7%；安寐汤高剂量组为6只,50%；安定对照组为4只,33.33%；七叶神安片对照组为4只,33.33%。安寐汤中剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组分别与正常对照组比较有统计学意义(P<0.05),安寐汤中剂量组,安寐汤高剂量组,安定对照组,七叶神安片对照组小鼠睡眠发生率明显高于正常对照组。
(四)安寐汤对抗士的宁致小鼠惊厥的作用研究
研究目的：探讨安寐汤对小鼠的抗惊厥作用。
研究方法：小鼠灌胃给药15天后,腹腔注射硝酸士的宁,记录小鼠惊厥数。结果进行统计学分析。
研究结果：正常对照组发生惊厥小鼠的数量为12只,发生率为100%；安寐汤低剂量组睡眠小鼠的数量为8只,发生率为66.7%；安寐汤中剂量组为3只,25.0%；安寐汤高剂量组为2只,16.7%；安定对照组为1只,8.3%；七叶神安片对照组为8只,66.7%。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组分别与正常对照组比较有统计学意义(P<0.05),安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组、七叶神安片对照组小鼠惊厥发生率明显低于正常对照组。
(五)安寐汤对失眠大鼠脑内中枢神经递质的影响
研究目的：检测安寐汤对失眠大鼠脑内5-HT、5-HIAA、NE、DA的含量的影响,探讨安寐汤改善失眠作用机制。
研究方法：实验用PCPA造成失眠大鼠模型,采用高效液相-电化学检测法检测失眠大鼠脑内5-HT、5-HIAA、NE、DA的含量变化。色谱条件为：色谱柱采用Phenomenex色谱柱(Luna C18,150×4.6mm,5μm); Alltech预柱(Alltima C185μm,Guard 7.5×4.6mm)。流动相采用乙腈：水相=13：87,用磷酸调水相的pH为4.0。流速为1.0ml／min。设定电压为+350mv。柱温为35℃。
研究结果：空白对照组与空白模型对照组比较有差异(P<0.05),空白模型对照组大鼠脑内5-HT含量显著降低。与空白模型对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组均能提高失眠大鼠脑内5-HT含量,其中安寐汤高剂量组、安定对照组能显著提高失眠大鼠脑内5-HT含量(P<0.05)。各组雌雄老鼠脑内5-HT含量的疗效比较,P>0.05,尚不能认为存在组内性别差异。
与空白对照组相比,空白模型对照组大鼠脑内5-HIAA含量显著降低(P<0.05)。与空白模型对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组均能提高失眠大鼠脑内5-HIAA含量,其中安寐汤高剂量组能显著提高失眠大鼠脑内5-HIAA含量(P<0.05)。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组间差异不明显。各组雌雄老鼠脑内5-HIAA含量的疗效比较,P>0.05,尚不能认为存在组内性别差异。
与空白对照组相比,空白模型对照组大鼠脑内NE含量有所升高。空白模型组大鼠脑内NE含量是较空白组有所增加,但两组相比无显著性差异。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组可降低空白模型对照组大鼠脑内升高的NE含量,但作用不显著。安寐汤高剂量组对失眠大鼠脑内NE含量的影响存在性别差异(P<0.05),雌老鼠脑内NE的含量低于雄老鼠。安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组间差异不明显。
与空白对照组相比,空白模型对照组大鼠脑内DA含量显著升高(P<0.05)。与空白模型对照组相比,安寐汤低剂量组、安寐汤中剂量组、安寐汤高剂量组、安定对照组均能降低失眠大鼠脑内DA含量,其中安寐汤中剂量组、安寐汤高剂量组能显著降低失眠大鼠脑内DA含量(P<0.05)。
第三部分研究结论
1.安寐汤对小鼠自主活动影响效果显著,可显著降低小鼠的自主活动性,说明安寐汤镇静效果明显,镇静作用优于安定和七叶神安片。
2.安寐汤对小鼠入睡潜伏期和睡眠时间影响效果显著,可显著缩短入睡潜伏期和延长睡眠时间,说明安寐汤催眠效果明显,催眠作用优于安定和七叶神安片。
3.安寐汤在提高小鼠睡眠发生率的作用上优于安定对照组和七叶神安片对照组,在降低小鼠惊厥发生率的作用上优于七叶神安片对照组,但比安定对照组作用效果弱。
4.安寐汤高、中、低不同剂量组之间的疗效对比结果表明,安寐汤各剂量组对小鼠自主活动影响效果以安寐汤中剂量组效果最为明显,对小鼠入睡潜伏期和睡眠时间影响效果以安寐汤低、中剂量组效果较为明显,对小鼠睡眠发生率作用以安寐汤中、高剂量组效果较为明显,对降低小鼠惊厥发生率作用以安寐汤中、高剂量组效果较为明显,提示中剂量可作为药物开发及临床用药的参考剂量。
5.安寐汤对不同性别老鼠镇静作用、催眠作用差异均不明显,提示安寐汤作用无性别上的差异。
6.通过对失眠大鼠脑内中枢神经递质含量的作用进行研究,我们发现安寐汤可以显著增加失眠大鼠脑内五羟色胺、五羟吲哚乙酸的含量,降低多巴胺的含量。安寐汤可能是通过增加五羟色胺、五羟吲哚乙酸的含量,降低多巴胺的含量而作用于睡眠—觉醒机制,以实现增加睡眠、改善睡眠的作用。
7.本实验采用高效液相-电化学检测法建立了快捷、准确、稳定的检测单胺类中枢神经递质含量的检测方法。结果表明,待测指标成分在10分钟内全部检测完成。各峰面积与神经递质浓度线性关系良好,各待测组分日内精密度良好,拟定的色谱条件下溶剂峰对样品各待测成分无干扰,各样品溶液中四个待测成分均能达到基线分离。
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 analysis
The 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 Study
Experiment I Anmeitang on the impact of the general behavior of mice
Objective: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 Anmeitang
Objective: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 hypnosisdose
Objective: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 mice
Objective: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 mice
Objective: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.
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