【作者】 黄凌；

【导师】 董志； 朱毅；

【作者基本信息】 重庆医科大学 ， 药理学， 2008， 硕士

【摘要】 帕金森病(Parkinson’s Disease,PD)是一种老年神经系统退行性疾病,PD患病率呈逐年上升趋势,在老年退行性疾病中发病率位居第二,仅次于阿尔茨海默病,发病率已达1.03%。目前临床所用的治疗PD药物数量少,且均有长期用药后疗效下降并出现严重不良反应等缺点,因此,开发高效低毒的PD防治药物显得尤为迫切。益智仁为姜科类植物益智(Alpinia Oxyphylla Miq.)的成熟干燥果实,是我国著名的四大南药之一。为了加快益智药材的开发,我国药学工作者对其进行了较系统的研究。研究发现,益智仁主要化学成分为挥发油类、微量元素、萜类、黄酮类及庚烷类等,其中挥发油占1%~2%,研究益智仁化学成分的重点为挥发油成分,用气质联用法已经分析出100余种化学成分,含量较高的有聚伞花烃、香橙醛、芳樟醇、桃金娘醛、α-蒎烯、β-蒎烯等,还含有锌、铜、铁、锰、钴、镁、钙等微量元素,其中锌、锰的含量远远高于其他补益药。现代医学研究表明,益智仁具有强心、抗癌、抗过敏、抗衰老、镇静、镇痛等多方面药理作用。近年来,以益智仁为主的方剂在益智健脑方面的临床应用越来越多,并且在动物实验中进一步证实益智仁具有良好的脑保护作用,可用于老年性痴呆及其他智能障碍类疾病的治疗,对神经细胞有抗氧化、抗衰老、减轻兴奋性毒性和抗凋亡作用。鉴于益智主产于海南岛,且现已大面积栽培,产量高、品质好。因此,充分利用益智进行深加工,研究益智仁挥发油对PD的作用,开发益智仁挥发油作为PD治疗药物很有必要,亦有特色。目的:研究益智仁挥发油对实验性PD的作用,并初步研究其作用机制。方法:(1)水蒸气法提取益智仁挥发油,采用气相色谱法分析实验过程中所用益智仁挥发油的化学成分,研究其可控性及稳定性;(2)采用小鼠BLISS法,对益智仁挥发油进行急性毒性研究,测定其LD50;(3)1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl- 1,2,3,6- tetrahydropyri-dine,MPTP)腹腔注射建立C57BL小鼠PD模型,对PD小鼠进行游泳、自主活动、水迷宫行为学实验,观察益智仁挥发油对MPTP所诱导PD的保护作用;(4)建立PD小鼠模型,溶剂提取法分离小鼠纹状体内DA、5-HT,层析法分离小鼠纹状体内HVA,荧光分光光度计法测定DA、5-HT、HVA含量,观察益智仁挥发油对小鼠脑内单胺类神经递质的保护作用;(5)紫外分光光度计法测定PD小鼠纹状体内SOD活力,MDA、GSH含量;(6)采用免疫组化法,分析益智仁挥发油对小鼠黑质致密部神经细胞TH、BCL-2及CASPASE-3蛋白表达的影响,采用TUNEL细胞凋亡技术分析益智仁对小鼠黑质致密部神经细胞凋亡的影响。结果:(1)益智仁挥发油主要化学成分稳定,质量可控;(2)BLISS法测得益智仁挥发油LD50为8.327ml·kg-1;(3)行为学实验结果显示益智仁挥发油能对抗MPTP所诱导的PD小鼠游泳时间缩短,并且缩短了小鼠游出水迷宫的潜伏期和错误次数,但对小鼠自主活动减少无明显影响;(4)预先给予益智仁挥发油可明显抑制MPTP所引起的小鼠脑组织内GSH含量下降和MDA生成增加,且能抑制纹状体SOD活力的下降;(5)益智仁挥发油能阻遏PD小鼠纹状体内单胺类神经递质下降,预先给予益智仁挥发油可明显抑制小鼠脑内DA、HVA、5-HT含量降低;(6)益智仁挥发油可增加PD小鼠黑质致密部(SNc)TH、BCL-2蛋白的表达,降低SNc内CASPASE-3表达,同时能抑制PD小鼠黑质致密部神经元细胞的凋亡。结论:(1)益智仁挥发油成分稳定、可控;(2)益智仁挥发油毒性甚微,发现益智仁挥发油能对抗PD所致的小鼠行为学改变;(3)通过对益智仁挥发油抗实验性帕金森病机制的初步研究,发现其抗PD机制可能与下列因素有关:①增强对氧自由基的清除能力,抑制脂质过氧化反应;②抑制MAO-B活性,阻遏PD小鼠脑内DA、HVA、5-HT分解代谢;③益智仁挥发油能增加自身抗氧能力,调节与细胞凋亡密切相关的BCL-2、CASPASE-3、TH等调控蛋白的含量和表达,抑制细胞凋亡。更多还原

【Abstract】 Parkinson’s Disease(PD) is a kind of gerontism nervous system degenerative disease.PD attack rate goes up year by year, which is second only to Alzheimer disease, and its disease incidence has already reached 1.03%.At present,there are few clinical drugs for PD treatment and there are shortcomings,such as curative effect decline and serious adverse effects after long-term administration,it is thus evident that it is important to development high efficiency low toxical drug for PD’s prevention and cure.Alpinia Oxyphylla Miq.fruit is mature and dry fruit, which is one of the most famous traditional medicine in South-China.To speed up the development of Alpinia Oxyphylla Miq.,Chinese pharmaceutical researchers have undertook systematic researches on AOF.Recent study results indicated that the main chemical compositions in Alpinia Oxyphylla Miq. fruit were essential oil, microelement, terpene flavonoids, heptanes, etc,and essential oil accounted for 1%～2%. As a result, the keypoint to research the chemical composition is to research essential oil.More than 100 kinds of chemical compositions has been analyzed by Gas chromatography mass spectrometry(GC-MS) method.The main compositions were p-cymene, neral,coriandrol, myrtenal,a-pinene,β-pinene, etc,and the main microele- ments were Zn,Cu,Fe,Mn,Gu,Mg,Ca etc.Zn and Mn were obviously higher than other tonic.Modern medicine study has shown that Alpinia Oxyphylla Miq.fruit have the pharmacologic action on strengthen heart power, anti- cancer,anti-anapyhlaxis, anti-aging, cons- tious-sedation and relief pain. Recent years, more and more Alpinia Oxyphylla Miq.fruit as primary composition in some compound preparation has been applicated on clinical researches for intelligence and brain.It has been proved that Alpinia Oxyphylla Miq.fruit has good effects on brain protection in animal experiment, and can be used to treat senile dementia and other disturbance of intelligence. It also has antioxygen, anti-ageing, anti-apoptosis effects and can lessen excitability toxicity effect on nerve cell.As far as it is concerned that Alpinia Oxyphylla Miq. fruit is mainly produced in Hainan Island, where Alpinia Oxyphylla Miq.has been cultivated in large areas with high output and fine quality, it is feasible to extract essential oil from AOF for PD treatment.Objective:To study the effects and mechanisms of AOF on the experimental PD.Methods:(1)Wet distillation method was used to extract Essential oil from AOF;vapor-phase chromatography was applied to analyse the p-Cymene of essential oil extracted from AOF for studying the stability and normallability of essential oil extracted from AOF; (2)BLISS method was used to study acute toxicity test in mice and determine the LD50 ;(3) The C57BL mice were formed with intraperitoneal injections of 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyri-dine(MPTP) to establish PD model; behavioral exhibition was explored by swimming test,water maze experiment and antonomic activities counting experiment;(4)Mice were formed with intraperitoneal injections of MPTP to establish PD model; the dopamine(DA), 5-hydroxytryptamine(5-HT) were abstracted by solvent extraction method; homovanillic acid(HVA) was abstracted by chroma- tographic analysis antigenic method;the DA,5-HT,HVA levels of striata were analysed by Fluorospectraphotometry method;observe essential oil extracted from AOF effect on monoamine neurotransmitter in mice brain; (5)The malondialdehyde(MDA),reduced glutathione hormone(GSH) and superoxide dismutase (SOD) activity from mice striatum were measured by ultraviolet spectrophotometer method to explore the mechanism of protect- ive effects;(6)The tyrosine hydroxylase(TH),B-cell leukemia- lymphoma- 2(BCL-2)and CASPASE-3 expressed in SNc were detected by Immunohisto- chemical SABC method;TUNEL staining method was used to detecte apoptosis in SNc of mice.Results:(1)The chemical compositions of essential oil extracted from AOF were stable and normallable; (2)The LD50 of essential oil extracted from AOF was 8.3269 ml·kg-1 and its 95% confident limit was 7.037-10.06 ml·kg-1,which suggests the toxicity of essential oil extracted from AOF might be minor;(3)Ethology experimental results showed that the AOF groups had significant difference with the MPTP model group;essential oil extracted from AOF could prolong swimming time of PD mice,shorten latency phase of mice in the water maze and decrease frequency of mice into blind-ending , but hadn’t significant effect on autonomic activities of PD mice;(4)Essential oil extracted from AOF obviously antagonized MPTP-induced elevation of MDA,as well as decreased of GSH level and SOD activity of brain tissue; (5)Essential oil extracted from AOF obviously antagonize MPTP-induced decrease of DA,HVA,5-HT of PD mice; (6)Essential oil extracted from AOF could significantly increase the TH,BCL-2 expression,obviously decrease CASPASE-3expression in SNc, meanwhile inhibit nerve cell Apoptosis in SNc.Conclusion:(1)The compositions of essential oil extracted from AOF are stable and normallable;(2)The toxicity of essential oil extracted from AOF is very low;essential oil extracted from AOF could antagonize ethology change of PD mice induced by MPTP,which suggests that AOF can have effects on prevention and cure PD induced by MPTP.(3)It is showed that the anti-Parkinson’s disease mechanisms of essential oil extracted from AOF might relate to the following factors. ①Essential oil extracted from AOF inhibits generation of oxygen- derived free radicals,and accelerates cleaning of free radicals,and relieve lipid peroxidation reaction, and maintains contents of SOD, GSH in normal level.②Essential oil extracted from AOF inhibits activity of monoamine oxidase-B(MAO-B), repress DA, 5-HT destructive metabolism.③Essential oil extracted from AOF could increase self-antioxygen capability, regulate the expression of modulin that intimate related to apoptosis such as BCL-2、CASEPASE-3、TH,etc.,then inhibits apoptosis.更多还原