摘要
目的建立单胺类神经递质多巴胺(DA)及去甲肾上腺素(NE)的高效液相色谱-电化学检测法(HPLC-ECD),探讨天麻乙酸乙酯萃取物对帕金森病(PD)大鼠脑组织中DA及NE含量的影响。方法(1)色谱条件:色谱柱:QU-3C18-15021型C;反相色谱柱(150 mm×2.1 mm);流动相为∶水相∶有机相(90∶10,V/V)[其中水相为磷酸二氢钾(0.076 mol/L),辛烷磺酸钠(25 mg/L),用磷酸调节pH至4.6;有机相为甲醇];流速:0.1 mL/min;柱温:28℃。电化学检测器工作电极:玻璃碳电极;参比电极:银/氯化银(Ag/AgCl)电极;检测电压:+0.7 V。(2)天麻乙酸乙酯萃取物对大鼠脑组织中神经递质含量的影响:选取健康成年SD大鼠,采用脑内定位注射6-羟基多巴胺(6-OHDA)建立PD大鼠模型。随机分为8组,即空白对照组、假手术组、模型对照组、阳性对照组(美多芭7.8 mg/kg)、吐温溶剂组、天麻乙酸乙酯萃取物低剂量组(5 g生药/kg)、中剂量组(10 g生药/kg)、高剂量组(20 g生药/kg)。各组灌胃给药治疗30 d。于末次给药2 h后分离取出大鼠脑组织,经处理后采用HPLC-ECD测定脑组织中神经递质-DA、NE的含量变化。结果(1)HPLC-ECD方法学考察结果表明,在本色谱条件下可以很好的分离单胺类递质DA、NE(R>1.5),且阴性对照(空白溶剂)在相应位置无干扰,线性关系、精密度、重复性、稳定性、加样回收率良好,可以用于DA、NE的检测;(2)样品检测结果表明,与模型组比较,天麻乙酸乙酯萃取物高剂量组(20 g生药/kg)能显著增加PD大鼠脑中DA的含量(P<0.05),天麻乙酸乙酯萃取物低、中、高剂量组(5、10、20 g生药/kg)对NE亦有明显增高作用(P<0.05)。结论采用HPLC-ECD方法进行单胺类神经递质DA、NE的检测,方法简便,结果准确;其检测结果表明,天麻乙酸乙酯萃取物能升高脑内单胺类神经递质的水平,显示出抗PD的作用。
Objective To establish a detection method of HPLC-ECD on DA and NE and explore the effect of Gastrodia ethyl acetate extract on the contents of DA and NE in Parkinson′s disease rats.Methods(1)Chromatographic conditions:Chromatography column was QU-3 C18-15021 C;reverse-phase chromatographic column(150 mm×2.1 mm).The mobile phase was water phase∶organic phase(90∶10,V/V).The water was potassium dihydrogen phosphate(0.076 mol/L)and 1-Octanesulfonic acid sodium salt solution(25 mg/L).The pH was adjusted to 4.6 with phosphoric acid.The organic phase was methanol.The flow rate was 0.1 mL/min.The column temperature was 28℃.The electrochemical detector working electrode was glassy carbon electrode.The reference electrode was silver/silver chloride(Ag/AgCl)electrode.The detection voltage was+0.7 V.(2)The effect of Gastrodia ethyl acetate extract on the neurotransmiter content of the brain tissues in rats:The healthy adult SD rats were selected and local injection of 6-hydroxydopamine(6-OHDA)was used to establish Parkinson′s disease rat model.The rats were randomly divided into 8 groups,namely control group,sham operation group,model group,positive group(medopa,7.8 mg/kg),Tween-80 group,Gastrodia ethyl acetate extract low dose group(5 g crude drug/kg),middle dose group(10 g crude drug/kg),high dose group(20 g crude drug/kg).Above groups were treated by intragastric administration for 30 days.And brain tissues were separated 2 hours after the last administration.After treatment,the contents of DA and NE were determined by HPLC-ECD.Results(1)HPLC-ECD methodological investigation results showed that under the chromatographic conditions,DA and NE can be well separated(R>1.5),and the negative control(blank solvent)had no interference at the corresponding position.Linear relationship,precision,repeatability,stability and sample recovery rate were good.It can be used for detecting the contents of DA and NE.(2)Compared with the model group,Gastrodia ethyl acetate extract high dose group(20 g crude drug/kg)significantly increased the content of DA in the brains of Parkinson′s disease rats(P<0.05).And the low dose group(5 g crude drug/kg),middle dose group(10 g crude drug/kg)and high dose group(20 g crude drug/kg)also increased the content of DA in the brains of Parkinson′s disease rats(P<0.05).Conclusion The HPLC-ECD method was used to detect the monoamine neurotransmitters DA and NE.The method is simple and the results are accurate and the results show that Gastrodia ethyl acetate extract can increase the levels of monoamine neurotransmitters in the brain,and it has the effect of anti-Parkinson′s disease.
作者
郭沛鑫
吴海妹
尹玉莲
解宇环
雷娜
GUO Peixin;WU Haimei;YIN Yulian;XIE Yuhuan;LEI Na(Yunnan University of Chinese Medicine,Kunming 650500,Yunnan,China)
出处
《辽宁中医杂志》
CAS
2021年第10期182-186,共5页
Liaoning Journal of Traditional Chinese Medicine
基金
国家自然科学基金(81660671)
云南省科学技术厅-云南中医药大学应用基础研究联合专项[2017FF116(-010)]
国家重点研发计划中医药现代化研究重点专项(2017YFC1703901)
作者简介
郭沛鑫(1976-),男,重庆人,副教授,博士,研究方向:中药及民族药;通讯作者:解宇环(1979-),女,云南昆明人,教授,博士,研究方向:中药药理学,E-mail:kmkamma@163.com;通讯作者:雷娜(1985-),女,云南昆明人,讲师,硕士,研究方向:中药药理学,E-mail:492078820@qq.com。
