天麻茎秆养生茶的研制及其抗惊厥作用研究

Study on the development of health tea with stalk of Gastrodia elata and its anticonvulsant effect

为合理利用长白山道地资源,以天麻茎秆为原料,与杨黄、青楷槭进行配伍,研制一款养生茶,并对其抗惊厥作用进行探究.在单因素实验基础上,采用正交实验设计、感官评分标准、高效液相色谱(High Performance Liquid Chromatography,HPLC)等方法对天麻茎秆养生茶的制备工艺进行优化;对其可溶性糖、总氨基酸和水浸出物的含量进行测定;应用气相色谱-质谱法(Gas Chromatography-mass Spectrometry,GC-MS)对其香气成分进行分析,建立指纹图谱;采用戊四氮(PTZ)和N-甲基-D-天冬氨酸(NMDA)建立两种小鼠惊厥模型,观察天麻茎秆养生茶(3000 mg/kg)对小鼠惊厥发作的行为影响,通过ELISA和免疫蛋白印迹分析(Western Blot)法检测小鼠脑中γ-氨基丁酸(GABA)、谷氨酸(Glu)、一氧化氮(NO)、五羟色胺(5-HT)、Na+-K+-ATP酶(Na+-K+-ATP)、天冬氨酸(Asp)、总抗氧化能力(AOC)、过氧化氢酶(CAT)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GSR)、丙二醛(MDA)、白介素1β(IL-1β)、白细胞介素10(IL-10)、肿瘤坏死因子α(TNF-α)以及凋亡蛋白Caspase-3、Bcl-2、Bax的含量变化;观察各组小鼠脑皮层及海马的病理学变化;免疫组织化学方法测定组织中c-Fos和TLR4蛋白的表达水平;并对天麻茎秆及养生茶的急性毒性进行了评价.结果显示:天麻茎秆养生茶的最佳工艺条件为:制天麻茎秆0.6 g,制杨黄0.1 g、青楷槭1.0 g,加沸水200 mL,冲泡时间10 min,冲泡次数为3.在此工艺条件下,测得可溶性糖量为39.4 mg/kg;总氨基酸量为3.2 mg/kg;冷、热浸法测得水浸出物的含量分别为391.6 g/kg、79.1 g/kg;建立了该养生茶的指纹图谱;且该养生茶可显著延长小鼠惊厥潜伏期、缩短惊厥时间、降低惊厥小鼠癫痫等级及死亡率,改善由PTZ、NMDA所致的脑内指标变化,减少神经元受损,降低脑皮层及海马组织中的c-Fos、TLR4蛋白表达水平;急性毒性结果表明,天麻茎秆及养生茶对小鼠的毒性极小,几乎无毒.天麻茎秆养生茶具有良好的抗惊厥作用;可以为合理开发利用这三种资源提供依据.

The stem of Gastrodia elata stalk which is compatible with Phellinus vaninii and Acer tegmentosum was developed a health tea,to explore its anticonvulsant effect,and make rational use of Changbai Mountain land resources.On the basis of single factor experiment,orthogonal preparation was designed,sensory scoring standard and High Performance Liquid Chromatography(HPLC)were used to optimize the preparation process of Gastrodia elata stalk health tea;the determination of soluble sugar,total amino acid and water extract content were carried out;Gas Chromatography-Mass Spectrometry(GC-MS)was used to analyze the aroma components and establish fingerprints.The two mouse convulsion models(Pentylenetetrazol(PTZ)and N-A)were established by basal-D-aspartate(NMDA)to observe the effect of Gastrodia elata stalk health tea(3000 mg/kg)on the behavior of seizure mice,Detectionγ-aminobutyric acid(GABA),glutamic acid(Glu),nitric oxide(NO),serotonin(5-HT),Na+-K+-ATPase(Na+-K+-ATP),aspartic acid(Asp),Total antioxidant capacity(AOC),catalase(CAT),glutathione(GSH),superoxide dismutase(SOD),glutathione reductase(GSR),malondialdehyde(MDA)),IL-1β,IL-10,IL-10,TNF-αand apoptosis proteins Caspase-3,Bcl-2,Bax of mouse brain by ELISA and Western Blot;and observed the cortex and sea of mice in each group Pathological changes;immunohistochemistry c-Fos and TLR4 protein expression levels were measured in tissue.The acute toxicity of Gastrodia elata stalk and health tea was evaluated.The results showed:The optimum conditions for the cultiva:tion of Gastrodia elata stalk health tea were 0.6 g of Gastrodia elata stalk,0.1 g of Phellinus vaninii,1.0 g of Acer tegmentosum,200 mL of boiling water,10 min of brewing time and 3 times of brewing.Under the conditions of this process,the amount of soluble sugar was 39.4 mg/kg;the total amino acid amount was 3.2 mg/kg;the contents of water extracts were 391.6 g/kg and 79.1 g/kg,respectively.The fingerprint of the health tea was established;and the health tea can significantly prolong the convulsion latency,shorten the convulsion time,reduce the level of epilepsy and mortality in convulsive mice,improve the changes of brain indicators caused by PTZ and NMDA,and reduce the nerves.Meta-damaged,reduced c-Fos,TLR4 protein expression levels in the cortex and hippocampus.The acute toxicity results showed that the Gastrodia elata stalk and health tea had little toxicity to mice and were almost non-toxic.Gastrodia elata stalk health tea has good anticonvulsant effect;it can provide a basis for rational development and utilization of these three resources.