大黄酚纳米囊、包合物、脂质体三种剂型的组织分布及药效学比较研究
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摘要
脑血管疾病（Cerebrovascular disease，CVD）是严重危害人类健康的疾病，其中以缺血性脑血管病（Ischemia cerebrovascular disease，ICVD）最为多见，是中老年人的常见病，具有高发病率、高患病率、高复发率、高致残率及高死亡率等特点，也是国内外重点防治的疾病之一。但由于其病因及发病机制尚未阐明，目前缺乏有效的治疗手段。因此，研究脑血管疾病的病因及发病机制、寻找有效的预防和治疗药物是国内外医药学领域的研究热点。近年来，国内外学者对应用天然药物有效成分治疗脑血管疾病进行了大量研究，但都未取得突破性进展。
大黄酚（Chrysophanol，Chry）是大黄中提取的蒽醌类化合物，我们前期的研究工作证实，大黄酚具有显著的抗衰老作用。但由于大黄酚单体在水中的溶解度低、性质不稳定、直接用药对胃肠有刺激作用等缺点，限制了其进一步的临床应用。前期的研究表明，配制大黄酚的溶剂主要是10 % N，N-二甲基甲酰胺（N，N-Dimethylformamide，DMF），但由于其为有机溶剂，具有一定毒性，只能低浓度、少量应用于动物实验研究，不能应用于临床。因此，为寻找适合临床应用的剂型，我们进行了大黄酚的临床用药剂型筛选研究。目前，本研究室已经完成了大黄酚聚氰基丙烯酸丁酯纳米囊、大黄酚羟丙基-β-环糊精包合物和大黄酚脂质体三种剂型制备工艺的研究，并分别进行了初步药效学和药代动力学考察。为进一步比较大黄酚三种剂型各自的特点，设立本课题。欲通过建立小鼠脑缺血再灌注损伤模型，分别给予大黄酚三种剂型药物，比较三种剂型不同剂量组的药效学作用及其组织分布特点，以期进一步完善三种制剂的药理学研究，同时筛选出最佳剂型应用于临床。
昆明种雄性小鼠285只，随机分为19组，分别为：①对照组：正常对照组、假手术组、脑缺血再灌注损伤模型组、DMF溶剂对照组、纳米囊对照组、包合物对照组、脂质体对照组；②大黄酚DMF（10 %DMF溶解）10.0，1.0，0.1 mg·kg-1三个剂量组；③大黄酚纳米囊10.0，1.0，0.1 mg·kg-1三个剂量组；④大黄酚包合物10.0，1.0，0.1 mg·kg-1三个剂量组；⑤大黄酚脂质体10.0，1.0，0.1 mg·kg-1三个剂量组。
除正常对照组和假手术组外，其余各组小鼠进行脑缺血再灌注操作，假手术组小鼠进行手术但不进行脑缺血再灌注操作。手术后，正常对照组、假手术组和模型对照组腹腔注射生理盐水，其余各组给予相应溶剂或药物，每天一次，连续给药18 d。给药d 8~d 9进行避暗实验，给药d 11~d 12进行跳台实验，给药d 14~d 15进行探索实验，给药d 17~d 18进行穿梭实验，通过实验观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠记忆障碍的改善作用。通过测定脑、肝、血清过氧化氢酶（Catalase，CAT）、谷胱甘肽过氧化物酶（Glutathioneperoxidase，GSH-Px）和超氧物歧化酶（Superoxide dismutase，SOD）、一氧化氮（Nitric oxide，NO）、一氧化氮合酶（Nitric oxide synthase，NOS）、单胺氧化酶-B（Monoamine oxidase-B，MAO-B）的活性；测定脑及肝丙二醛（Malondialdehyde，MDA）、脑及心脏组织脂褐素（Lipofuscin，LF）的含量；观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠上述各氧化相关酶活性和代谢产物含量的影响。通过测定脑组织乙酰胆碱酯酶（Acetylcholinesterase，AChE）的活性，观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠AChE活性的影响。通过测定脑组织氨基酸（Amino acid，AA）的含量，观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠脑内氨基酸的影响。通过观察小鼠断头耐缺氧生存时间、张口呼吸次数和脑指数，比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠耐缺氧能力的影响。进行脑、肝组织2，3，5-三苯基氯化四氮唑（2，3，5-Triphenyltetrazolium chloride，TTC）染色和苏木精-伊红（Hematoxylin and eosin，HE）染色，观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠脑梗死面积和病理形态学的影响。连续腹腔给药18 d后将小鼠断头处死，取血并迅速留取各组织器官（心、脑、肝、脾、肺、肾），通过高效液相色谱法（Highperformance liquid chromatography，HPLC）测定各组织中大黄酚含量，观察不同剂型大黄酚的组织分布差异。
1三种大黄酚剂型的药效学实验
1.1三种大黄酚剂型对学习记忆的影响
结果显示，脑缺血再灌注损伤可引起小鼠明显的记忆功能障碍（p<0.05~p<0.01）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组均可不同程度地改善脑缺血再灌注损伤所致记忆功能障碍（p<0.05~p<0.01），且呈剂量依赖性：在避暗实验中，小鼠进入暗室的潜伏期延长，错误次数明显减少（p<0.05~p<0.01）；在跳台实验中，小鼠在平台上的潜伏期和错误次数减少（p<0.05~p<0.01）；在探索实验中，小鼠进入暗室潜伏期明显延长、爬高时间和到达明室时间均明显提前，爬高次数显著增多（p<0.05~p<0.01）；在穿梭实验中，小鼠被电击次数和电击时间明显较少，主动逃避时间延长（p<0.05）。而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物这两种剂型改善记忆障碍作用与大黄酚DMF组相比，无显著性差异（p>0.05）；而大黄酚脂质体10.0 mg·kg-1剂量组改善作用差异显著（p<0.01）。
结果表明，大黄酚各剂型均具有改善脑缺血再灌注损伤作用，其中大黄酚脂质体作用最佳。
1.2三种大黄酚剂型对组织氧化相关酶活性和代谢产物含量的影响
结果显示，脑缺血再灌注损伤可引起脑、肝、血清CAT、GSH-Px、SOD活性降低（p<0.05~p<0.01），使NO、NOS和MAO-B活性升高（p<0.05~p<0.01）；使脑及肝组织MDA含量升高（p<0.01）、脑及心脏组织LF含量增高（p<0.01）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组可不同程度改善脑缺血再灌注损伤引起的氧化相关酶活性和代谢产物含量的变化（p<0.05~p<0.01），呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物这两种剂型与大黄酚DMF组相比作用相似，无显著性差异（p>0.05）；而大黄酚脂质体的作用较好，大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均具有改善脑缺血再灌注损伤，缓解损伤对氧化相关酶活性和代谢产物含量的影响，其中大黄酚脂质体作用最佳。
1.3三种大黄酚剂型对脑组织乙酰胆碱酯酶活性的影响
结果显示，脑缺血再灌注损伤可引起脑组织AChE活性升高（p<0.01）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组均可不同程度抑制脑组织中AChE的活性（p<0.05~p<0.01），且呈剂量相关性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对AChE活性的影响与大黄酚DMF组无显著性差异（p>0.05）；而大黄酚脂质体对AChE的作用较好，其中以大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均可抑制组织中AChE活性，其中大黄酚脂质体作用最佳。
1.4三种大黄酚剂型对脑内氨基酸含量的影响
结果显示，脑缺血再灌注损伤可引起脑组织AA含量显著降低（p<0.05）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组可明显增加AA的含量（p<0.05~p<0.01），且呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对脑内氨基酸含量的影响与大黄酚DMF组无显著性差异（p>0.05）；而大黄酚脂质体对脑内氨基酸的作用较好，其中以大黄酚脂质体10.0mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均可不同程度影响脑内氨基酸水平，其中大黄酚脂质体作用最佳。
1.5三种大黄酚剂型的耐缺氧能力的影响
结果显示，脑缺血再灌注损伤可引起小鼠断头耐缺氧生存时间和张口呼吸次数明显减少（p<0.05），脑指数减小（p<0.05）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组小鼠断头耐缺氧生存时间明显延长、张口呼吸次数明显增加（p<0.05），脑指数增大（p<0.05），且呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对耐缺氧能力的影响与大黄酚DMF组无显著性差异（p>0.05）；而大黄酚脂质体对耐缺氧的作用较好，其中以大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.05）。
结果表明，大黄酚各剂型均可不同程度增强脑耐缺氧能力，其中大黄酚脂质体作用最佳。
1.6三种大黄酚剂型对脑、肝组织病理形态的影响
结果显示，脑缺血再灌注损伤可引起脑、肝组织病理形态的改变。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0mg·kg-1剂量组脑梗死体积减小，具有显著性差异（p<0.01）；海马区出现神经元密集，结构正常，神经细胞间隙变小，神经细胞数量增多，神经元间质界限清晰；肝小叶结构完整，肝细胞排列规则，数目增多，且呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无明显改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对脑缺血再灌注损伤所致脑、肝组织形态的病理变化无改善作用（p>0.05）；而大黄酚脂质体对脑缺血再灌注损伤的作用较好，其中以大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均可不同程度缓解脑缺血再灌注损伤小鼠的病理形态改变，其中大黄酚脂质体作用最佳。
2三种大黄酚剂型的组织分布
结果显示，大黄酚在小鼠体内的组织分布含量依次为：血>脑>肾>肝>心>脾>肺，大黄酚三种制剂10.0，1.0，0.1 mg·kg-1剂量组与大黄酚DMF组比较，血、肝及脑组织中的大黄酚含量均高于大黄酚组DMF组，有显著性差异（p<0.05~p<0.01），且呈剂量依赖性。
各剂型之间组织分布情况比较，相同剂量的大黄酚纳米囊和包合物两种剂型在组织中的分布与大黄酚组比较，无显著性差异（p>0.05）；而大黄酚脂质体在各组织中含量较高，其中大黄酚脂质体10.0 mg·kg-1剂量组测得各组织含量最高（p<0.01）。
结果表明，大黄酚DMF及三种剂型均可使大黄酚分布于各组织器官，且组织含量有明显差异，以血和脑组织分布较多；剂型中以大黄酚脂质体分布含量较高。
上述研究结果表明，脑缺血再灌注损伤可建立血管性痴呆动物模型，通过学习记忆行为学实验、氧化相关酶活性及代谢产物含量测定、断头耐缺氧实验以及脑梗死面积和病理形态学观察等实验进行了药效学研究。结果证实，三种大黄酚剂型具有改善脑缺血再灌注损伤的作用，通过降低组织中MDA、LF含量及NO、NOS和MAO-B活性，提高CAT、GSH-Px、SOD活性，进而组织的抗氧化能力增强；抑制组织中AChE活性，进而使胆碱能神经功能增强；调节脑内氨基酸水平，从而减轻神经元的损伤；增强脑组织的耐缺氧能力，改善脑缺血再灌注的损伤作用。大黄酚聚氰基丙烯酸丁酯纳米囊、大黄酚羟丙基-β-环糊精包合物、大黄酚脂质体剂型之间的组织分布相比较，以大黄酚脂质体在各组织的分布较好，药效作用也最为显著。大黄酚脂质体有望成为大黄酚临床应用的新剂型，特别是各种途径注射给药的剂型，应用于临床治疗缺血性脑血管疾病。
Cerebral vascular diseases are harmful to human life and heath.Ischemia cerebrovascular disease (ICVD) is a common disease inmiddle-aged and elderly people. Its characteristic includes high diseaseincidence rate，high prevalence rate，high recurrence rate，high cripplingrate and high mortality rate. ICVD is one of key diseases that have beenprevented or treated at home and abroad. So a lot of human and materialresources have been put into the basic and clinical study. Up to now theprimary pathological mechanisms and causes of ICVD have not beendefinite. As the lack of effective diagnostic and therapeutic methods，it isvery important and urgent to study the drug for ICVD. In recent years，inorder to find better ways and better drugs，domestic and foreignresearchers have done a lot of researches in treating ICVD with the activeingredients of natural medicines，but there is no satisfactory therapy forICVD.
Chrysophanol is the compound from rhubarb anthraquinones. Theprevious study confirms that it has an evident anti-aging effect. As thesolubility of chrysophanol in water is low，its nature is unstable and thestimulating effect on gastrointestinal is obvious when used directly，itsfurther clinical application is limited. In previous studies，chrysophanol’ssolvent is mainly 10 % N, N-dimethylformamide (DMF). Because it isorganic solvent and has harmful effect，it can’t be used in clinicaladministration. But it can be used in the experiment at low concentrationand small dosage. In order to find new and suitable dosage forms forclinical application，we conducted screening study on clinical pharmacology research of chrysophanol formulations. Currently，chrysophanol was made into nanocapsules loading with chrysophanol bypolybutylcyanoacrylate，chrysophanol hydroxypropyl-β-cyclodextrininclusion complex and chrysophanol liposomes by the best preparationmethod and technics，and their preliminary pharmacokinetic studies werecompleted. In order to screen out the best formulation for clinicalapplications，we compared the differences of organization distribution andpharmacodynamics effects in three chrysophanol formulations by adoptingto cerebral ischemia reperfusion.
285 male mice were divided into nineteen groups randomly：Group 1were normal control group，sham-operated group，cerebral ischemiareperfusion model control group，and the solvent control group of DMF，nanocapsules，inclusion complex and liposomes；Group 2 werechrysophanol (10 % DMF dissolved) 10.0，1.0，0.1 mg·kg-1 dose group；Group 3 were nanocapsules loading with chrysophanol by polybutylcyanoacrylate10.0，1.0，0.1 mg·kg-1 dose group；Group 4 were chrysophanolhydroxypropyl-β-cyclodextrin inclusion complex 10.0，1.0，0.1 mg·kg-1dose group；Group 5 were chrysophanol liposomes 10.0，1.0，0.1 mg·kg-1dose group.
In various groups，cerebral ischemia reperfusion were administeredexcept normal control and sham-operated group. Sham group wasoperated on but not executed cerebral ischemia reperfusion. Aftermodeling，corresponding drugs were administered for every groups andnormal saline (NS) for normal group，sham-operated group，cerebralischemia reperfusion model control group，once a day for eighteen days inthe same way. The ethological experiments，about learning and memory，such as passive avoidance test (d 8~d 9)，exploratory movement test (d11~d 12)，step-through and step-down test (d 14~d 15) and shuttle test (d17~d 18) etc，were observed the improving effects of chrysophanolformulations on memory impairment cerebral ischemia reperfusion-induced. Through determination of the activities of CAT，GSH-Px，SOD，NO，NOS，MAO-B in brain，liver and blood tissues，the contents of MDA in brain and liver tissues，the contents of LF in brainand heart tissues，we observed the effects on oxidative related enzymes’activity and metabolites’content of chrysophanol formulations. Throughdetermination of activity of AChE in brain tissue，we observed thepharmacodynamic effects of chrysophanol formulations. Throughdetermination of contents of amino acid(AA) in brain tissue，we observedthe pharmacodynamic effects of chrysophanol formulations. Throughdetermination of survival time in beheaded hypoxia mice and brain index，we observed the pharmacodynamic effects of chrysophanol formulations.Through the TTC and HE dying experiment in brain and liver tissues，weobserved the brain’s infarction area and pathomorphological changes ofchrysophanol formulations. Through determination of chrysophanolcontent in tissues by High Performance Liquid Chromatography(HPLC)，we observed tissue distribution’s differences of chrysophanolformulations.
1 Pharmacodynamicharmacodynamics experiments of three chrysophanol formulations
1.1 Effect of three chrysophanol formulations on learning andmemory
Results showed that cerebral ischemia could cause significantmemory impairment in mice (p<0.05~p<0.01). Compared with thecorresponding model group，chrysophanol DMF and chrysophanolformulations (10.0，1.0 mg·kg-1 dose groups) could improve theimpairments of memory with cerebral ischemia reperfusion：instep-through test，the latency had an extended tendency，and the number ofentry times was significantly reduced (p<0.05~p<0.01)；In exploratorymovement test，the time of climbing darkroom was extended，height andbright room was evidently brought forward and the number of climbingheight was evidently increased (p<0.05~p<0.01)；In platform test，the latency and the number of error times were significantly decreased(p<0.05~p<0.01)；In shuttle test，the number and time of electric shockwas significantly decreased，the time of initiative avoiding was evidentlyprolonged (p<0.05). Chrysophanol DMF and chrysophanol formulations(0.1 mg·kg-1 dose group) had no significant improvements in the aboveindicators (p>0.05).
Compared with chrysophanol group，the two groups of nanocapsulesand inclusion complex had no significant difference in improving memoryimpairment；However，chrysophanol liposome was better in this respect，particularly in 10.0 mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving theimpairments of memory with cerebral ischemia reperfusion and the bestformulation was chrysophanol liposomes.
1.2 Effect of three chrysophanol formulations on oxidative relatedenzymes’activity and metabolites’content in tissues
Results showed that cerebral ischemia-reperfusion injury could lowerthe levels of CAT，GSH-Px，SOD in brain，liver and blood tissues(p<0.05~p<0.01)，improve the levels of NO，NOS and MAO-B in brain，liver and blood tissues (p<0.05~p<0.01)，increase the contents of MDA inbrain and liver tissues (p<0.01) and the contents of LF in brain and hearttissues (p<0.01). Compared with the corresponding model group，chrysophanol DMF and chrysophanol formulations (10.0，1.0 mg·kg-1dose groups) could improve the levels of oxidative related enzymes’activity and metabolites’content (p<0.05~p<0.01)，which could improvethe cerebral ischemia-reperfusion injury，and these results were related tothe dose. Chrysophanol DMF and chrysophanol formulations (0.1 mg·kg-1dose group) had no significant improvements to the above indicators.Compared with chrysophanol DMF group，the nanocapsules and inclusioncomplex groups had no significant difference (p>0.05)；However， chrysophanol liposome was better in this respect，particularly in 10.0mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving the levels ofoxidative related enzymes’activity and metabolites’content and the bestformulation was chrysophanol liposomes.
1.3 Effect of three chrysophanol formulations on AChE in brain tissue
Results showed that，cerebral ischemia-reperfusion injury couldimprove the activity of AChE in brain tissue (p<0.01). Compared with thecorresponding model group，chrysophanol DMF and chrysophanolformulations (10.0，1.0 mg·kg-1 dose groups) could lower the activity ofAChE (p<0.05~p<0.01)，and the results were related to the dose；Compared with chrysophanol DMF group，the two groups of nanocapsulesand inclusion complex had no significant difference in the activity ofAChE (p>0.05)；However，chrysophanol liposome was better in thisrespect，particularly in 10.0 mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving the activity ofAChE and the best formulation was chrysophanol liposomes.
1.4 Effect of three chrysophanol formulations on amino acid in braintissue
Results showed that，cerebral ischemia-reperfusion injury couldlower the contents of AA in brain tissue (p<0.05). Compared with thecorresponding model group，chrysophanol DMF and chrysophanolformulations (10.0，1.0 mg·kg-1 dose groups) could improve the contentsof AA in brain tissue (p<0.05~p<0.01)，and the results were related to thedose；Compared with chrysophanol DMF group，the two groups ofnanocapsules and inclusion complex had no significant difference in thecontents of AA (p>0.05)；However，chrysophanol liposome was better inthis respect，particularly in 10.0 mg·kg-1 dose group (p<0.01). These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the contentsof AA in brain tissue and the best formulation was chrysophanolliposomes.
1.5 Effect of three chrysophanol formulations on hypoxia tolerance
Results showed that，cerebral ischemia-reperfusion injury couldshorten the survival time of beheaded hypoxia，the number of mouth andbrain index (P<0.05). Compared with the corresponding model group，chrysophanol DMF and chrysophanol formulations (10.0，1.0 mg·kg-1dose groups) could prolong the survival time of beheaded hypoxia，thenumber of mouth and brain indexed (P<0.05)，and the result were relatedto the dose；Compared with chrysophanol DMF group，the two groups ofnanocapsules and inclusion complex had no significant difference(p>0.05)；However，chrysophanol liposome was better in this respect，particularly in 10.0 mg·kg-1 dose group (p<0.05).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the effects ofhypoxia tolerance and the best formulation was chrysophanol liposomes.
1.6 Effect of three chrysophanol formulations on pathomorphology inbrain and liver tissues
Results were as follows，cerebral ischemia reperfusion injury haddifferent levels of pathological changes in brain and liver tissues. Incontrast to the corresponding model group，chrysophanol DMF andchrysophanol formulations (10.0，1.0 mg·kg-1 dose groups) could decreasecerebral infarction size (P<0.01)，neurons’density and neuronal structureof nerve cells in hippocampal area were normal，the interspace among thenerve cells were decreased，the number of nerve cells increased and thecircumscription between cell nucleus and membrane of some nerve cellswere clear，lobule boundary，hepatic cell structure and arrangement ofwere also normal，the number of hepatic cells increased，and the results were related to the dose；In contrast to chrysophanol DMF group，the twogroups of nanocapsules and inclusion complex had no significantdifference (p>0.05)；However，chrysophanol liposome was better in theserespects，particularly in 10.0 mg·kg-1 dose group(p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the effects ofthe pathological form and the best formulation was chrysophanolliposomes.
2 TissuTissue distribution of three chrysophanol formulations
Results showed that，the order of chrysophanol concentration inmice’s tissues by HPLC：blood>brain>kidney>liver>heart>spleen>lungs，compared with chrysophanol DMF，chrysophanol concentration ofchrysophanol formulations (10.0，1.0，0.1 mg·kg-1 dose groups) in brain，liver and blood tissues were a significant difference (p<0.05~p<0.01)，andthe results were related to the dose；Compared with chrysophanol DMFgroup，the two groups of nanocapsules and inclusion complex had nosignificant difference (p>0.05)；However，chrysophanol liposome wasbetter in this respect，particularly in 10.0 mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the tissuedistribution，among them，the tissue distribution of blood and brain washigher and the best formulation was chrysophanol liposomes.
In summary，the cerebral ischemia reperfusion could result insenescence. The learning and memory experiments，antioxidant enzymeassays，beheaded hypoxia，cerebral infarction size and pathologicalmorphous experiments had been studied. The concentrations of chry in thedifferent tissue were determined by HPLC. All the results implied thatchrysophanol formulations could lower the contents of MDA，LF and theactivities of NO，NOS，MAO-B，improve the activities of CAT，GSH-Px，SOD，inhibit the activity of AChE，affect the level amino acid in brain， enhance the effect of hypoxia tolerance，and improve the pathologyprocess of disease. Among these chrysophanol formulations，chrysophanolliposomes was best，so it might become one of new drugs of treatingICVD in the future，in particular，the injectable dosage forms can beadministered in various routes.
大黄酚（Chrysophanol，Chry）是大黄中提取的蒽醌类化合物，我们前期的研究工作证实，大黄酚具有显著的抗衰老作用。但由于大黄酚单体在水中的溶解度低、性质不稳定、直接用药对胃肠有刺激作用等缺点，限制了其进一步的临床应用。前期的研究表明，配制大黄酚的溶剂主要是10 % N，N-二甲基甲酰胺（N，N-Dimethylformamide，DMF），但由于其为有机溶剂，具有一定毒性，只能低浓度、少量应用于动物实验研究，不能应用于临床。因此，为寻找适合临床应用的剂型，我们进行了大黄酚的临床用药剂型筛选研究。目前，本研究室已经完成了大黄酚聚氰基丙烯酸丁酯纳米囊、大黄酚羟丙基-β-环糊精包合物和大黄酚脂质体三种剂型制备工艺的研究，并分别进行了初步药效学和药代动力学考察。为进一步比较大黄酚三种剂型各自的特点，设立本课题。欲通过建立小鼠脑缺血再灌注损伤模型，分别给予大黄酚三种剂型药物，比较三种剂型不同剂量组的药效学作用及其组织分布特点，以期进一步完善三种制剂的药理学研究，同时筛选出最佳剂型应用于临床。
昆明种雄性小鼠285只，随机分为19组，分别为：①对照组：正常对照组、假手术组、脑缺血再灌注损伤模型组、DMF溶剂对照组、纳米囊对照组、包合物对照组、脂质体对照组；②大黄酚DMF（10 %DMF溶解）10.0，1.0，0.1 mg·kg-1三个剂量组；③大黄酚纳米囊10.0，1.0，0.1 mg·kg-1三个剂量组；④大黄酚包合物10.0，1.0，0.1 mg·kg-1三个剂量组；⑤大黄酚脂质体10.0，1.0，0.1 mg·kg-1三个剂量组。
除正常对照组和假手术组外，其余各组小鼠进行脑缺血再灌注操作，假手术组小鼠进行手术但不进行脑缺血再灌注操作。手术后，正常对照组、假手术组和模型对照组腹腔注射生理盐水，其余各组给予相应溶剂或药物，每天一次，连续给药18 d。给药d 8~d 9进行避暗实验，给药d 11~d 12进行跳台实验，给药d 14~d 15进行探索实验，给药d 17~d 18进行穿梭实验，通过实验观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠记忆障碍的改善作用。通过测定脑、肝、血清过氧化氢酶（Catalase，CAT）、谷胱甘肽过氧化物酶（Glutathioneperoxidase，GSH-Px）和超氧物歧化酶（Superoxide dismutase，SOD）、一氧化氮（Nitric oxide，NO）、一氧化氮合酶（Nitric oxide synthase，NOS）、单胺氧化酶-B（Monoamine oxidase-B，MAO-B）的活性；测定脑及肝丙二醛（Malondialdehyde，MDA）、脑及心脏组织脂褐素（Lipofuscin，LF）的含量；观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠上述各氧化相关酶活性和代谢产物含量的影响。通过测定脑组织乙酰胆碱酯酶（Acetylcholinesterase，AChE）的活性，观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠AChE活性的影响。通过测定脑组织氨基酸（Amino acid，AA）的含量，观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠脑内氨基酸的影响。通过观察小鼠断头耐缺氧生存时间、张口呼吸次数和脑指数，比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠耐缺氧能力的影响。进行脑、肝组织2，3，5-三苯基氯化四氮唑（2，3，5-Triphenyltetrazolium chloride，TTC）染色和苏木精-伊红（Hematoxylin and eosin，HE）染色，观察比较三种大黄酚制剂对脑缺血再灌注损伤模型小鼠脑梗死面积和病理形态学的影响。连续腹腔给药18 d后将小鼠断头处死，取血并迅速留取各组织器官（心、脑、肝、脾、肺、肾），通过高效液相色谱法（Highperformance liquid chromatography，HPLC）测定各组织中大黄酚含量，观察不同剂型大黄酚的组织分布差异。
1三种大黄酚剂型的药效学实验
1.1三种大黄酚剂型对学习记忆的影响
结果显示，脑缺血再灌注损伤可引起小鼠明显的记忆功能障碍（p<0.05~p<0.01）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组均可不同程度地改善脑缺血再灌注损伤所致记忆功能障碍（p<0.05~p<0.01），且呈剂量依赖性：在避暗实验中，小鼠进入暗室的潜伏期延长，错误次数明显减少（p<0.05~p<0.01）；在跳台实验中，小鼠在平台上的潜伏期和错误次数减少（p<0.05~p<0.01）；在探索实验中，小鼠进入暗室潜伏期明显延长、爬高时间和到达明室时间均明显提前，爬高次数显著增多（p<0.05~p<0.01）；在穿梭实验中，小鼠被电击次数和电击时间明显较少，主动逃避时间延长（p<0.05）。而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物这两种剂型改善记忆障碍作用与大黄酚DMF组相比，无显著性差异（p>0.05）；而大黄酚脂质体10.0 mg·kg-1剂量组改善作用差异显著（p<0.01）。
结果表明，大黄酚各剂型均具有改善脑缺血再灌注损伤作用，其中大黄酚脂质体作用最佳。
1.2三种大黄酚剂型对组织氧化相关酶活性和代谢产物含量的影响
结果显示，脑缺血再灌注损伤可引起脑、肝、血清CAT、GSH-Px、SOD活性降低（p<0.05~p<0.01），使NO、NOS和MAO-B活性升高（p<0.05~p<0.01）；使脑及肝组织MDA含量升高（p<0.01）、脑及心脏组织LF含量增高（p<0.01）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组可不同程度改善脑缺血再灌注损伤引起的氧化相关酶活性和代谢产物含量的变化（p<0.05~p<0.01），呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物这两种剂型与大黄酚DMF组相比作用相似，无显著性差异（p>0.05）；而大黄酚脂质体的作用较好，大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均具有改善脑缺血再灌注损伤，缓解损伤对氧化相关酶活性和代谢产物含量的影响，其中大黄酚脂质体作用最佳。
1.3三种大黄酚剂型对脑组织乙酰胆碱酯酶活性的影响
结果显示，脑缺血再灌注损伤可引起脑组织AChE活性升高（p<0.01）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组均可不同程度抑制脑组织中AChE的活性（p<0.05~p<0.01），且呈剂量相关性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对AChE活性的影响与大黄酚DMF组无显著性差异（p>0.05）；而大黄酚脂质体对AChE的作用较好，其中以大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均可抑制组织中AChE活性，其中大黄酚脂质体作用最佳。
1.4三种大黄酚剂型对脑内氨基酸含量的影响
结果显示，脑缺血再灌注损伤可引起脑组织AA含量显著降低（p<0.05）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组可明显增加AA的含量（p<0.05~p<0.01），且呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无显著改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对脑内氨基酸含量的影响与大黄酚DMF组无显著性差异（p>0.05）；而大黄酚脂质体对脑内氨基酸的作用较好，其中以大黄酚脂质体10.0mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均可不同程度影响脑内氨基酸水平，其中大黄酚脂质体作用最佳。
1.5三种大黄酚剂型的耐缺氧能力的影响
结果显示，脑缺血再灌注损伤可引起小鼠断头耐缺氧生存时间和张口呼吸次数明显减少（p<0.05），脑指数减小（p<0.05）。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0 mg·kg-1剂量组小鼠断头耐缺氧生存时间明显延长、张口呼吸次数明显增加（p<0.05），脑指数增大（p<0.05），且呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对耐缺氧能力的影响与大黄酚DMF组无显著性差异（p>0.05）；而大黄酚脂质体对耐缺氧的作用较好，其中以大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.05）。
结果表明，大黄酚各剂型均可不同程度增强脑耐缺氧能力，其中大黄酚脂质体作用最佳。
1.6三种大黄酚剂型对脑、肝组织病理形态的影响
结果显示，脑缺血再灌注损伤可引起脑、肝组织病理形态的改变。与各对应的模型组比较，大黄酚DMF及三种不同剂型的10.0，1.0mg·kg-1剂量组脑梗死体积减小，具有显著性差异（p<0.01）；海马区出现神经元密集，结构正常，神经细胞间隙变小，神经细胞数量增多，神经元间质界限清晰；肝小叶结构完整，肝细胞排列规则，数目增多，且呈剂量依赖性；而大黄酚DMF及三种不同剂型的0.1 mg·kg-1剂量组对上述各项指标无明显改善作用（p>0.05）。
各剂型之间药效作用比较，大黄酚纳米囊和包合物两种剂型对脑缺血再灌注损伤所致脑、肝组织形态的病理变化无改善作用（p>0.05）；而大黄酚脂质体对脑缺血再灌注损伤的作用较好，其中以大黄酚脂质体10.0 mg·kg-1剂量组改善作用较显著（p<0.01）。
结果表明，大黄酚各剂型均可不同程度缓解脑缺血再灌注损伤小鼠的病理形态改变，其中大黄酚脂质体作用最佳。
2三种大黄酚剂型的组织分布
结果显示，大黄酚在小鼠体内的组织分布含量依次为：血>脑>肾>肝>心>脾>肺，大黄酚三种制剂10.0，1.0，0.1 mg·kg-1剂量组与大黄酚DMF组比较，血、肝及脑组织中的大黄酚含量均高于大黄酚组DMF组，有显著性差异（p<0.05~p<0.01），且呈剂量依赖性。
各剂型之间组织分布情况比较，相同剂量的大黄酚纳米囊和包合物两种剂型在组织中的分布与大黄酚组比较，无显著性差异（p>0.05）；而大黄酚脂质体在各组织中含量较高，其中大黄酚脂质体10.0 mg·kg-1剂量组测得各组织含量最高（p<0.01）。
结果表明，大黄酚DMF及三种剂型均可使大黄酚分布于各组织器官，且组织含量有明显差异，以血和脑组织分布较多；剂型中以大黄酚脂质体分布含量较高。
上述研究结果表明，脑缺血再灌注损伤可建立血管性痴呆动物模型，通过学习记忆行为学实验、氧化相关酶活性及代谢产物含量测定、断头耐缺氧实验以及脑梗死面积和病理形态学观察等实验进行了药效学研究。结果证实，三种大黄酚剂型具有改善脑缺血再灌注损伤的作用，通过降低组织中MDA、LF含量及NO、NOS和MAO-B活性，提高CAT、GSH-Px、SOD活性，进而组织的抗氧化能力增强；抑制组织中AChE活性，进而使胆碱能神经功能增强；调节脑内氨基酸水平，从而减轻神经元的损伤；增强脑组织的耐缺氧能力，改善脑缺血再灌注的损伤作用。大黄酚聚氰基丙烯酸丁酯纳米囊、大黄酚羟丙基-β-环糊精包合物、大黄酚脂质体剂型之间的组织分布相比较，以大黄酚脂质体在各组织的分布较好，药效作用也最为显著。大黄酚脂质体有望成为大黄酚临床应用的新剂型，特别是各种途径注射给药的剂型，应用于临床治疗缺血性脑血管疾病。
Cerebral vascular diseases are harmful to human life and heath.Ischemia cerebrovascular disease (ICVD) is a common disease inmiddle-aged and elderly people. Its characteristic includes high diseaseincidence rate，high prevalence rate，high recurrence rate，high cripplingrate and high mortality rate. ICVD is one of key diseases that have beenprevented or treated at home and abroad. So a lot of human and materialresources have been put into the basic and clinical study. Up to now theprimary pathological mechanisms and causes of ICVD have not beendefinite. As the lack of effective diagnostic and therapeutic methods，it isvery important and urgent to study the drug for ICVD. In recent years，inorder to find better ways and better drugs，domestic and foreignresearchers have done a lot of researches in treating ICVD with the activeingredients of natural medicines，but there is no satisfactory therapy forICVD.
Chrysophanol is the compound from rhubarb anthraquinones. Theprevious study confirms that it has an evident anti-aging effect. As thesolubility of chrysophanol in water is low，its nature is unstable and thestimulating effect on gastrointestinal is obvious when used directly，itsfurther clinical application is limited. In previous studies，chrysophanol’ssolvent is mainly 10 % N, N-dimethylformamide (DMF). Because it isorganic solvent and has harmful effect，it can’t be used in clinicaladministration. But it can be used in the experiment at low concentrationand small dosage. In order to find new and suitable dosage forms forclinical application，we conducted screening study on clinical pharmacology research of chrysophanol formulations. Currently，chrysophanol was made into nanocapsules loading with chrysophanol bypolybutylcyanoacrylate，chrysophanol hydroxypropyl-β-cyclodextrininclusion complex and chrysophanol liposomes by the best preparationmethod and technics，and their preliminary pharmacokinetic studies werecompleted. In order to screen out the best formulation for clinicalapplications，we compared the differences of organization distribution andpharmacodynamics effects in three chrysophanol formulations by adoptingto cerebral ischemia reperfusion.
285 male mice were divided into nineteen groups randomly：Group 1were normal control group，sham-operated group，cerebral ischemiareperfusion model control group，and the solvent control group of DMF，nanocapsules，inclusion complex and liposomes；Group 2 werechrysophanol (10 % DMF dissolved) 10.0，1.0，0.1 mg·kg-1 dose group；Group 3 were nanocapsules loading with chrysophanol by polybutylcyanoacrylate10.0，1.0，0.1 mg·kg-1 dose group；Group 4 were chrysophanolhydroxypropyl-β-cyclodextrin inclusion complex 10.0，1.0，0.1 mg·kg-1dose group；Group 5 were chrysophanol liposomes 10.0，1.0，0.1 mg·kg-1dose group.
In various groups，cerebral ischemia reperfusion were administeredexcept normal control and sham-operated group. Sham group wasoperated on but not executed cerebral ischemia reperfusion. Aftermodeling，corresponding drugs were administered for every groups andnormal saline (NS) for normal group，sham-operated group，cerebralischemia reperfusion model control group，once a day for eighteen days inthe same way. The ethological experiments，about learning and memory，such as passive avoidance test (d 8~d 9)，exploratory movement test (d11~d 12)，step-through and step-down test (d 14~d 15) and shuttle test (d17~d 18) etc，were observed the improving effects of chrysophanolformulations on memory impairment cerebral ischemia reperfusion-induced. Through determination of the activities of CAT，GSH-Px，SOD，NO，NOS，MAO-B in brain，liver and blood tissues，the contents of MDA in brain and liver tissues，the contents of LF in brainand heart tissues，we observed the effects on oxidative related enzymes’activity and metabolites’content of chrysophanol formulations. Throughdetermination of activity of AChE in brain tissue，we observed thepharmacodynamic effects of chrysophanol formulations. Throughdetermination of contents of amino acid(AA) in brain tissue，we observedthe pharmacodynamic effects of chrysophanol formulations. Throughdetermination of survival time in beheaded hypoxia mice and brain index，we observed the pharmacodynamic effects of chrysophanol formulations.Through the TTC and HE dying experiment in brain and liver tissues，weobserved the brain’s infarction area and pathomorphological changes ofchrysophanol formulations. Through determination of chrysophanolcontent in tissues by High Performance Liquid Chromatography(HPLC)，we observed tissue distribution’s differences of chrysophanolformulations.
1 Pharmacodynamicharmacodynamics experiments of three chrysophanol formulations
1.1 Effect of three chrysophanol formulations on learning andmemory
Results showed that cerebral ischemia could cause significantmemory impairment in mice (p<0.05~p<0.01). Compared with thecorresponding model group，chrysophanol DMF and chrysophanolformulations (10.0，1.0 mg·kg-1 dose groups) could improve theimpairments of memory with cerebral ischemia reperfusion：instep-through test，the latency had an extended tendency，and the number ofentry times was significantly reduced (p<0.05~p<0.01)；In exploratorymovement test，the time of climbing darkroom was extended，height andbright room was evidently brought forward and the number of climbingheight was evidently increased (p<0.05~p<0.01)；In platform test，the latency and the number of error times were significantly decreased(p<0.05~p<0.01)；In shuttle test，the number and time of electric shockwas significantly decreased，the time of initiative avoiding was evidentlyprolonged (p<0.05). Chrysophanol DMF and chrysophanol formulations(0.1 mg·kg-1 dose group) had no significant improvements in the aboveindicators (p>0.05).
Compared with chrysophanol group，the two groups of nanocapsulesand inclusion complex had no significant difference in improving memoryimpairment；However，chrysophanol liposome was better in this respect，particularly in 10.0 mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving theimpairments of memory with cerebral ischemia reperfusion and the bestformulation was chrysophanol liposomes.
1.2 Effect of three chrysophanol formulations on oxidative relatedenzymes’activity and metabolites’content in tissues
Results showed that cerebral ischemia-reperfusion injury could lowerthe levels of CAT，GSH-Px，SOD in brain，liver and blood tissues(p<0.05~p<0.01)，improve the levels of NO，NOS and MAO-B in brain，liver and blood tissues (p<0.05~p<0.01)，increase the contents of MDA inbrain and liver tissues (p<0.01) and the contents of LF in brain and hearttissues (p<0.01). Compared with the corresponding model group，chrysophanol DMF and chrysophanol formulations (10.0，1.0 mg·kg-1dose groups) could improve the levels of oxidative related enzymes’activity and metabolites’content (p<0.05~p<0.01)，which could improvethe cerebral ischemia-reperfusion injury，and these results were related tothe dose. Chrysophanol DMF and chrysophanol formulations (0.1 mg·kg-1dose group) had no significant improvements to the above indicators.Compared with chrysophanol DMF group，the nanocapsules and inclusioncomplex groups had no significant difference (p>0.05)；However， chrysophanol liposome was better in this respect，particularly in 10.0mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving the levels ofoxidative related enzymes’activity and metabolites’content and the bestformulation was chrysophanol liposomes.
1.3 Effect of three chrysophanol formulations on AChE in brain tissue
Results showed that，cerebral ischemia-reperfusion injury couldimprove the activity of AChE in brain tissue (p<0.01). Compared with thecorresponding model group，chrysophanol DMF and chrysophanolformulations (10.0，1.0 mg·kg-1 dose groups) could lower the activity ofAChE (p<0.05~p<0.01)，and the results were related to the dose；Compared with chrysophanol DMF group，the two groups of nanocapsulesand inclusion complex had no significant difference in the activity ofAChE (p>0.05)；However，chrysophanol liposome was better in thisrespect，particularly in 10.0 mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in improving the activity ofAChE and the best formulation was chrysophanol liposomes.
1.4 Effect of three chrysophanol formulations on amino acid in braintissue
Results showed that，cerebral ischemia-reperfusion injury couldlower the contents of AA in brain tissue (p<0.05). Compared with thecorresponding model group，chrysophanol DMF and chrysophanolformulations (10.0，1.0 mg·kg-1 dose groups) could improve the contentsof AA in brain tissue (p<0.05~p<0.01)，and the results were related to thedose；Compared with chrysophanol DMF group，the two groups ofnanocapsules and inclusion complex had no significant difference in thecontents of AA (p>0.05)；However，chrysophanol liposome was better inthis respect，particularly in 10.0 mg·kg-1 dose group (p<0.01). These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the contentsof AA in brain tissue and the best formulation was chrysophanolliposomes.
1.5 Effect of three chrysophanol formulations on hypoxia tolerance
Results showed that，cerebral ischemia-reperfusion injury couldshorten the survival time of beheaded hypoxia，the number of mouth andbrain index (P<0.05). Compared with the corresponding model group，chrysophanol DMF and chrysophanol formulations (10.0，1.0 mg·kg-1dose groups) could prolong the survival time of beheaded hypoxia，thenumber of mouth and brain indexed (P<0.05)，and the result were relatedto the dose；Compared with chrysophanol DMF group，the two groups ofnanocapsules and inclusion complex had no significant difference(p>0.05)；However，chrysophanol liposome was better in this respect，particularly in 10.0 mg·kg-1 dose group (p<0.05).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the effects ofhypoxia tolerance and the best formulation was chrysophanol liposomes.
1.6 Effect of three chrysophanol formulations on pathomorphology inbrain and liver tissues
Results were as follows，cerebral ischemia reperfusion injury haddifferent levels of pathological changes in brain and liver tissues. Incontrast to the corresponding model group，chrysophanol DMF andchrysophanol formulations (10.0，1.0 mg·kg-1 dose groups) could decreasecerebral infarction size (P<0.01)，neurons’density and neuronal structureof nerve cells in hippocampal area were normal，the interspace among thenerve cells were decreased，the number of nerve cells increased and thecircumscription between cell nucleus and membrane of some nerve cellswere clear，lobule boundary，hepatic cell structure and arrangement ofwere also normal，the number of hepatic cells increased，and the results were related to the dose；In contrast to chrysophanol DMF group，the twogroups of nanocapsules and inclusion complex had no significantdifference (p>0.05)；However，chrysophanol liposome was better in theserespects，particularly in 10.0 mg·kg-1 dose group(p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the effects ofthe pathological form and the best formulation was chrysophanolliposomes.
2 TissuTissue distribution of three chrysophanol formulations
Results showed that，the order of chrysophanol concentration inmice’s tissues by HPLC：blood>brain>kidney>liver>heart>spleen>lungs，compared with chrysophanol DMF，chrysophanol concentration ofchrysophanol formulations (10.0，1.0，0.1 mg·kg-1 dose groups) in brain，liver and blood tissues were a significant difference (p<0.05~p<0.01)，andthe results were related to the dose；Compared with chrysophanol DMFgroup，the two groups of nanocapsules and inclusion complex had nosignificant difference (p>0.05)；However，chrysophanol liposome wasbetter in this respect，particularly in 10.0 mg·kg-1 dose group (p<0.01).
These results demonstrated that chrysophanol DMF andchrysophanol formulations had different levels in influencing the tissuedistribution，among them，the tissue distribution of blood and brain washigher and the best formulation was chrysophanol liposomes.
In summary，the cerebral ischemia reperfusion could result insenescence. The learning and memory experiments，antioxidant enzymeassays，beheaded hypoxia，cerebral infarction size and pathologicalmorphous experiments had been studied. The concentrations of chry in thedifferent tissue were determined by HPLC. All the results implied thatchrysophanol formulations could lower the contents of MDA，LF and theactivities of NO，NOS，MAO-B，improve the activities of CAT，GSH-Px，SOD，inhibit the activity of AChE，affect the level amino acid in brain， enhance the effect of hypoxia tolerance，and improve the pathologyprocess of disease. Among these chrysophanol formulations，chrysophanolliposomes was best，so it might become one of new drugs of treatingICVD in the future，in particular，the injectable dosage forms can beadministered in various routes.
引文
1何惠美.脑血管疾病患者的心理评估和护理[J].中国当代医药，2011，18(1)：113-114.
2王耀辉，张路，韩嫣然.脑血管疾病康复护理[J].吉林医学，2011，32(2)：407-408.
3陈跃红，王宝丽.脑血管疾病患者生活质量调查[J].慢性病学杂志，2010，12(10)：1368.
4奥俊青.脑血管疾病的心理护理[J].中国现代药物应用，2011，5(1)：188.
5 Bordet R，Ouk T，Onténiente B，et al. Cerebral ischaemia：tomorrow's therapeutictracks[J]. Med Sci(Paris)，2009，25(10)：847-854.
6王树，薛贵平，张丹参，等.大黄酚对脑缺血再灌注小鼠学习记忆障碍及耐缺氧的影响[J].陕西医学杂志，2008，37(4)：402-404.
7张丹参，张力，薛贵平，等.大黄酚的抗衰老作用[J].中国医院药学杂志，2005，25(1)：15-17.
8王树，张丹参，张力，等.大黄酚对脑缺血再灌注小鼠记忆功能的保护作用[J].中国老年学杂志，2009，29(15)：1934-1936.
9王树，张力，张丹参，等.大黄酚对脑缺血再灌注小鼠探索功能的影响[J].时珍国医国药，2007，18(12)：3011-3013.
10董晓华，张丹参.大黄酚对Aβ25-35所致AD大鼠学习记忆及LTP的影响[J].中国药理学通报，2009，25(5)：682-685.
11刘久青，李茂星.大黄游离蒽醌固体分散体的制备及溶出研究[J].中国医药指南，2010，8(4)：35-37.
12黄红娜，张丹参，郑晓霞，等.大黄酚聚氰基丙烯酸丁酯纳米囊的制备工艺及质量研究[J].中草药，2010，41(4)：547-550.
13 Koffi A A，Agnely F，Ponchel G，et al. Modulation of the rheological andmucoadhesive properties of thermosensitive poloxamer-based hydrogels intendedfor the rectal administration of quinine[J]. European Journal of PharmaceuticalSciences，2006，27(4)：328-335.
14 Mura P，Maestrelli F，Gonzalez-Rodriguez ML，et al. Development，characterization and in vivo evaluation of benzocaine-loaded liposomes[J]. Eur JPharm Biopharm，2007，67(1)：86-95.
15 Himori N，Watanabe H，Akaike N，et al. Cerebral ischemia model with consciousmice：involvement of NMDA receptor activation and derangement of learning andmemory ability[J]. Pharmacol Meth，1990，23：311-327.
16 MeCann SK，DustingG J，Roulston C L. Early increase of Nox4 NADPH oxidaseand super oxide generation following endothelin-1-induced stroke in consciousrats[J]. JNeurosciRes，2008，86(11)：2524-2534.
17 Terao Y，OhtaH，Oda A，et al. Macrophage inflammatory protein-3alpha plays a keyrole in the inflammatory cascade in rat focal cerebral ischemia[J]. Neurosci Res，2009，64(1)：75-82.
18 DaiH B，FuQ L，ShenY，et al. The histamineH3 receptor antagonist clobenpropitenhancesGABA release to protect againstNMDA-induced excitotoxicity throughthe cAMP/protein kinase A pathway in cultured cortical neurons[J].EurJPharmacol，2007，563(1/3)：117-123.
19李春颖，曲娴，李东盈.反复脑缺血再灌注对小鼠学习记忆和脑内氧化损伤的影响[J].北华大学学报，2009，10(4)：322-326.
20 St-Pierre J，Buckinghan JA，Roebuck SJ，et al. Topology of superoxide productionfrom different sites in the mitochondrial electron transport chain[J]. J Biol Chem，2002，277(47)：44784-44790.
21 Devi SA，Kiran TR. Regional responses in antioxidant system to exercise trainingand dietary Vitamin E in aging rat brain[J]. Neurobiol Aging，2004，25：501-508.
22 Grey AD. The reductive hotspot hypothesis of mammalian aging：membranemetabolism magnifies mutant mitochondrial mischief[J]. Eur J Biochem，2002，269(8)：2003.
23王树，张丹参，薛贵平，等.大黄酚对脑缺血再灌注小鼠脑组织H2O2和CAT的影响[J].中药药理与临床，2008，24(4)：22-24.
24 Sandhu SK，Kaur G. Alterations in oxidative stress scavenger system in aging ratbrain and Iumphocytes[J]. Biogerontology.2002，3(3)：161-173.
25 Giardino R，Giavaresi G，Fini M，et al. The role of different ehemical modificationsof superoxide dismutase in preventing a prolonged muscular ischemis/reperfusioninjury[J]. Artif Cells Blood Immubiol Biotechmol.2002，30(3)：189-198.
26况时祥，刘继刚，肖燕，等.脑通胶囊对血管性痴呆大鼠学习记忆能力及脑组织NO、SOD含量的影响[J].中国中医急症，2010，19(8)：1368-1370.
27蔡建伟，吴颢昕，晋光荣，等.双根清脑煎剂对血管性痴呆大鼠模型脑海马组织及血清SOD MDA的影响[J].中华中医药学刊，2007，25(6)：1167-1168.
28杨文明，王时光，洪亮，等.智脑胶囊对血管性痴呆模型大鼠行为学及脑组织SOD活性、MDA含量的影响[J].中西医结合心脑血管病杂志，2007，5(9)：833-835.
29张会珍，李文丽，马小顺，等.电针对拟血管性痴呆小鼠SOD和MDA的影响[J].中国中医基础医学杂志，2010，16(11)：1034-1035.
30顾饶胜，王艳春，刘微，等.大豆异黄酮对急性衰老小鼠学习记忆障碍的改善作用[J].吉林医药学院学报，2007，28(3)：125-127.
31刘秋芳，逯晓波，靳翠红，等.碱性电解水对D-半乳糖致小鼠血液和肝、肾、脑组织脂质过氧化作用的影响[J].环境与健康杂志，2010，27(2)：105-107.
32张晓杰，张静艳，赵坤，等.通络救脑口服液对AD大鼠海马单胺氧化酶活性、脂褐素含量的影响[J].中国老年学杂志，2010，30，(18)：2634-2636.
33薛茜，邹玉安，韩敏，等.康脑液对脑缺血再灌注损伤大鼠血清及脑组织匀浆SOD、MDA、NO的影响[J].中西医结合心脑血管病杂志，2010，8(6)：709-710.
34 Tsai SK，Hung LM，Fu YT，et al. Resveratrol neuroprotective effects during focalcerebral ischemia injury via nitric oxidemechanism in rats[J]. JVasc Surg，2007，46(2)：346-353.
35 Anctil M，Poulain I，Pelletier C. Nitric oxide modulates peristaltic muscle activityassociated with fluid circulation in the sea pansy Renilla koellikeri[J]. JExp Biol，2005，208(10)：2005-2017.
36熊文欣，李文峰.硫化氢对脑缺血再灌注损伤大鼠血清及脑组织NO和NOS的影响[J].现代中西医结合杂志，2010，19(16)：1971-1972.
37袁野，熊缨，杨俊卿，等.单胺氧化酶B在神经元退行性变模型中的意义研究[J].重庆医科大学学报，2006，31(5)：681-683，714.
38姚五湖，龙斌，周日笑.贯叶连翘提取物提高衰老小鼠学习记忆能力的研究[J].今日药学，2010，20(4)：37-39.
39戴恩来，马鸿斌，欧秀梅，等.通络益智散对拟血管性痴呆大鼠AChE，MDA和SOD的影响[J].甘肃中医，2006，19(5)：38-40.
40苗艳艳，苗明三，马霄，等.五味子醇提部位对反复脑缺血再灌注模型小鼠脑能量代谢的影响[J].中华中医药杂志，2009，24(9)：1207-1209.
41杨牧祥，武常生，于文涛，等.醒脑启智胶囊对血管性痴呆小鼠脑组织海马区乙酰胆碱酯酶的影响[J].北京中医药大学学报，2006，29(3)：177-180.
42刘杰，华赟鹏，谭敏谊，等.芍药苷注射液对脑缺血-再灌注沙土鼠脑组织中兴奋性氨基酸及腺苷含量的影响[J].中药材，2010，33(9)：1456-1460.
43张新春，吕光耀，秦秀德.益气活血中药对大鼠缺血-再灌注脑组织中氨基酸含量的影响[J].中国医药导报，2009，6(22)：30-32.
44陆晖，吴云虎，陆艳玲，等.酸枣仁皂苷A对脑缺血再灌注损伤大鼠神经行为学及脑组织氨基酸含量的影响[J].广西中医药，2008，31(6)：47-50.
45颜天华，贾莹，杨伟，等.红景天苷对大鼠局灶性脑缺血/再灌注损伤的保护作用[J].中国药理学通报，2008，24(11)：1521-1524.
46 Liu F，Schafer DP，McCullough LD. TTC，fluoro-Jade B and NeuN stainingconfirm evolving phases of infarction induced by middle cerebral arteryocclusion[J]. J Neurosci Methods，2009，179(1)：1-8.
47王红艳，李威，孙博谦，等.全脑缺血再灌注小鼠额叶、海马区神经元损伤的病理研究[J].中国老年学杂志，2006，26(8)：1088-1090.
48 Yu J，Novgorodov SA，Chudakova D，et al. JNK3 signaling pathway activatesceramide synthase leading to mitochondrial dysfunction[J]. J Biol Chem，2007，3：1-24.
49张兴毅，宋国林，韩江全，等.全脑缺血再灌注后大鼠海马回中Bcl-2及Bax的表达[J].西部医学，2009，21(11)：1850-1852.
50 Yang XF，He W，Lu WH，et al. Effects of seutellarin on liver funetion after brainischemia/reperfusion in rats[J]. Acta Pharmacol Sin，2003，24(11)：1118-1124.
1 Fang Y Z，Yang S，Wu G Y. Free Radicals，Antioxidants，and Nutrition[J].Nutrition，2002，18(10)：872.
2 Cai Y Z，Luo Q，Sun M. Antioxidant Activity and Phenolic compounds of 112Traditional Chinese Medicinal Plants Associated with Anticancer[J]. Life Sci，2004，74：2157-2184.
3 Gerhardt G，Cass W，Yi A，et al. Changes in somatodendritic bur not terminaldopamine regulation in rhesus monkeys[J]. J Neurochem，2002，80(1)：168-177.
4 Siles E.Martinez-Lara E，Canuedo A，et al. Age-related changes of the nitric oxidesystem in the rat brain[J]. Brain Res，2002，956(2)：385-392.
5 Goncharova ND，Lapin BA，Khavinson VKh. Age-associated endocrinedysfunctions and approaches to their correction[J]. Bull Exp Biol Med，2002，134(5)：417-421.
6 Trazona R，Solana R，Ouyang Q，et al. Basic biology and clinical impact ofimmunosenescence[J]. Exp Gerontol 2002，37：183.
7 Tarazona R，Solana R，Ouyang Q，et al. Basic biology and elinical impact ofimmunosenescence[J]. Exp Gerontol，2002，37：183.
8房林，赵振民.皮肤衰老机制的研究进展[J].人民军医，2010，53(2)：149-152.
9 Etzev DE，Lolov SR，Usunoff KG. Aging and synaptic changes in theparaventricular hypothalamic nucleus of the rat[J]. Acta Physiol Pharmacol Bulg，2003，27(2-3)：75-82.
10 Wu G，Yan S. Analysis of distributions of amino acids in the primary structure ofapoptosis regulator Bcl-2 family according to the random mechanism[J]. JBiochem Mol Biol Biophys，2002，6(6)：407-414.
11 Takeuchi A，Mishina Y，Miyaishi O，et al. Heterozygosity with respect to Zfp148causes complete loss of fetal germ cells during mouse embryogenesis[J]. NatGenet，2003，33(2)：172-176.
12杨茂林，孟思进.线粒体氧化损伤在衰老发生机制中的作用[J].医学综述，2010，16(9)：1297-1300.
13张丹丹，白云燕.衰老分子生物学研究进展[J].黑龙江医药科学，2008，31(6)：82-83.
14高凌云，李国栋，童坦君.延缓衰老相关的小分子物质研究进展[J].生物化学与生物物理进展，2010，37(9)：932-938.
15 Brunori M，Luciano P，Gilson E，et al. The telomerase cycle：normol andpathological aspects[J]. J Mol Med，2005，83(4)：244～257.
16彭艳华，王跃.细胞衰老与机体老化关系的研究进展[J].中国老年学杂志，2008，28(9)：932-934.
17 Zhang ZC，Schmitt MT，Mumford JL. Effects of arsenic on telomerase andtelomeres in relation to cell proliferation and apoptosis in human keratinocytes andleukemia cells in vitro[J]. Carcinogenesis，2003，24(11)：1811-1817.
18 Phaneuf S，Leeuwenburgh C. Cytochrome c release from mitochondria in the agingheart：a possible mechanism for apoptosis with age[J]. Am J Physol Regul IntegrComp Physol，2002，282(2)：R423-430.
19古彦铮，强亦忠，倪祥庭，等.干细胞的衰老学说[J].中国组织工程研究与临床康复2008，12(34)：6731-6734.
20彭立伟，来吉祥，何聪芬，等.非酶糖基化与皮肤衰老的研究进展[J].中国老年学杂志，2010，30(20)：3027-3029.
21 Stadtman ER，Levine RL. Free radical-mediated oxidation of free amino acids andamino acid residues in proteins[J]. Amino Acids，2003，25(3-4)：207-218.
22周文丽，张建鹏，冯伟华，等.脑衰老机制与脑疾病的关系[J].生命的化学，2008，28(4)：435-438.
23于海忠，赵淑敏，葛志华. D-半乳糖衰老大鼠下颌下腺超微结构的改变[J].河北医药，2009，31(11)：1287-1288.
24雷秀娟，冯凯，孙立伟，等.人参皂苷抗衰老机制的研究进展[J].氨基酸和生物资源，2010，32(1)：44-47.
25杨吉平，赖红，方欣，等.人参皂苷Rb1对阿尔茨海默病大鼠海马结构β-淀粉样蛋白表达的影响[J].中国组织化学与细胞化学杂志，2008，17(4)：301-305.
26彭书玲，郭兆安.三七总皂苷的作用机制研究进展[J].中国中西医结合急救杂志，2008，15(1)：63-64.
27齐艳萍，李和平，陈雪龙.鹿茸药理作用的研究进展[J].经济动物学报，2008，12(1)：54-55.
28 Hongping Pan，Li Gao. Protecting Mechanism of Puerarin on the Brain Neurocyteof Rat in Acute Local Ischemia Brain Injury and Local CerebralIschemia-reperfusion Injury[J]. Journal of the Pharmaceutical Society of Japan，2008，128(11)：1689-1698.
29 Li Gao，Xunming Ji，Juexian Song，et al. Puerarin protects against ischemic braininjury in a rat model of transient focal ischemia[J]. Neurological Research，2009，31(4)：402-406.
30 Xingsan Tang，Yazhen Ma. Protective effects of Breviscapine injection against focalcerebral ischemia-reperfusion injury in rats[J]. Chinese Journal of ClinicalRehabilitation，2005，9(29)：243-245.
31钟振国，吴登攀，王进声，等.三七总皂苷对老年性痴呆动物模型快速老化小鼠大脑Aβ1-40、Aβ1-42表达的影响[J].中药材，2009，32(1)：82-85.
32 Tombaugh GC，Rowe WB，Chow AR，et al. Theta-frequency syna-pticpotentiationin CA1 invito distinguishes cognitively impaired from unimpaired aged Fischer
344 rats[J]. Neuro-sci，2002，22(22)：9932-9940.
33刘霞，包翠芬，魏嘉，等.人参皂苷Rg1对脑缺血-再灌注大鼠海马CA1区神经元的保护作用及机制[J].解剖科学进展，2010，16(2)：177-180.
34陈超杰，张文生.七治疗阿尔茨海默氏病的研究进展[J].时珍国医国药，2008，19(7)：1559-1564.
35李维祖，李卫平，尹艳艳，等.黄芪总苷及黄芪甲苷对糖皮质激素诱导老前期小鼠记忆障碍的保护作用及其机制[J].安徽医药，2008，12(7)：592-594.
36 Chan RYK，ChenWF，DongA，et al. Estrogen-like activity of Ginsenoside Rg1derived fromPanax notoginseng[J]. J Clin Endocrinol Metab，2006，87(8)：3691-3695.
37 Byung SK，Tae SK，Cheorl HK. Salviae miltiorrhizae radix inhibits superoxidegeneration by activated rat microglias and mimics the action of amphetamine on invitro rat striatal dopamine release[J]. Neurochem Res，2004，29(10)：1837-1845.
38钟桂书，何渊民，廖勇梅.雷公藤、人参与芳维甲酸乙酯对培养光老化真皮成纤维细胞的影响[J].中国组织工程研究与临床康复，2010，14(2)：267-271.
39周燕，宁宗，田磊，等.三七总皂苷对大鼠海马CA1区兴奋性突触活动的影响[J].广西医科大学学报，2010，27(4)：532-535.
40 Sparg S G，Light M E，Staden J V. Biological Activities and Distribution of PlantSaponins[J]. J Ethnopharm，2004，94：219-243.
41栗平.何首乌抗衰药理与临床应用[J].慢性病学杂志，2010，12(10)：1271-1272.
42李娜，贾芳.人参皂苷治疗阿尔茨海默病作用机制的研究进展[J].医学综述，2008，14(17)：2691-2693.
43 Tian J，Fu F，Geng M，et al. Neuroprotective effect of 20(S)-ginsenoside Rg3 oncerebral ischemia in rats[J]. Neurosci Lett，2005，374(2)：92-97.
44 Jeong S M，Lee J H，Kim J H，et al. Stereospecificity of ginsenoside Rg3 action onion channels[J]. Mol Cells，2004，18(3)：245-251.
45 Hua Li，Changqing Deng，Beiyang Chen，et al. Total saponins of Panax notoginsengmodulate the expression of caspases and attenuate apoptosis in rats following focalcerebral ischemia-reperfusion [J]. Journal of Ethnopharmacology，2009，121(3)：412-418.
46王东吉，尚改萍，武凡，等.三七皂苷单体Rg1对大鼠脑缺血再灌注损伤保护作用的研究[J].中国中医基础医学杂志，2008，14(7)：513-515.
47刘婕，许浚，张铁军.苯丙素苷类化合物防治神经退行性疾病的研究进展[J].药物评价研究，2009，32(1)：62-66.
48 Zhanjun Zhang，Runguo Wu，Pengtao Li，et al. Baicalin administration is effectivein positive regulation of twenty-four ischemia/reperfusion-related proteinsidentified by a proteomic study[J]. Neurochemistry International，2009，54(8)：488-496.
49张凤普.人参皂苷的作用机理研究[J].临床合理用药，2010，3(21)：158.
50 Ji XY，Tan Benny Kg，Zhu YC，et al. Comparison of cardioprotective effects usingramipril and DanShen for the treatment of acute myocardial infarction in rats[J].Life Sci，2003，73：1413-1426.
51 Ikeda K，Negishi H，Yamori Y. Antioxidant nutrients and hypoxia/ischemia braininjury in rodents[J]. Toxicology，2003，189(1-2)：55-61.
52 Chandrasekaran K，Mehrabian Z，Spinnewyn B，et al. Bilobailide，a componentof theGinkgo bilobaextract (EGB761)，protects against neural death in global brainischemia and in glutamate-induced excitotoxicity[J]. Cell Mol Biol，2002，48(6)：663-669.
53 Giuseppe G，Peter J O. Potential Toxicity of Flavonoids and other DietaryPhenolics：Significance for their Chemopreventive and Anticancer Properties[J].Free Rad Bio Med，2004，37(3)：287-303.
54 Clarkson AN，Liu H，Pearson L，et al. Neuroprotective effects of sperminefollowing hypoxia-ischemia-induced braidamage：a mechanistic study[J]. FASEBJ，2004，18(10)：1114-1116.
55 Wei I H，Wu Y C，Wen C Y，et al. Green tea polypheno(-)-epigallocatechin gallateattenuates the neuronal NADPH d/nNOS expression in the nodose ganglion ofhypoxic rat[J]. Brain Res，2004，999(1)：73-80.
56谢甦，李丽红，李丽.三七总皂苷抗衰老的实验研究[J].世界中西医结合杂志，2008，3(2)：86-88.
57 Wen J X，Mo Y，Tai F F，et al. Partial neuroprotective effect of pretreatment withtanshinone IIA on neonatal hypoxiaischemia brain damage[J]. Pediatr Res，2005，58(4)：784-790.
58薛亮，范英昌，李庆雯，等.丹酚酸B对离体培养内皮祖细胞VEGF、bFGFmRNA表达及抗氧化酶活性的影响[J].中国老年学杂志，2011，31(2)：236-238.
59祝飞虹，高根德.中药治疗老年性痴呆临床研究近况[J].实用中医药杂志，2011，27(1)：68-69.
60杜娟，陈虹.肉苁蓉及其有效成分对脑损伤的保护作用研究进展[J].时珍国医国药，2010，21(9)：2333-2334.
61 Toyokuni S，Tanaka T，Kawaguchi N. R，et al. Effects of the phenolic contents ofmauritianendemic plantextracts on promoter activities of antioxidant enzymes[J].Free Radic Res. 2003，37：1215.
62 Hyang D G，Kwang H L，Hyoung J K，et al. Neuroprotective effects ofantioxidative flavonoids，quercetin(+)-dihydroquercetin and quercetin 3-methylether isolated from Opuntia ficus-indica Var saboten[J]. Brain Res，2003，965(1-2)：130-136.
63 Choi E J，Lee BH，Chee KM. Long-term combined administration of quercetin anddaidzein inhibits quercetin-induced suppression of glutathione antioxidantdefenses[J]. Food ChemToxicol，2005，43(5)：793-798.
64 GronebergD.A，Grosse-Siestrup C and FischerA. In vitromodels to studyhepatotoxicity[J]. ToxicolPalhol，2002，30：394.
65 Zbarsky V，Datla K P，Parkar S，et al. Neuroprotective properties of the naturalphenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a
6-OHDA model of Parkinson’s disease[J]. Free Radic Res，2005，39(10)：1119-1125.
66 Luo Y，Qin Z，Hong Z，et al. Astragaloside IV protects against ischemic brain injuryin a murine model of transient focal ischemia[J]. Neurosci Lett，2004，363(3)：218-223.
67 Wei X B，Liu H Q，Sun X，et al. Hydroxysafflor yellow A protects rat brains againstischemia-reperfusion injury by antioxidant action[J]. Neurosci Lett，2005，386(1)：58-62.
68 Tatiana D，Galina T，Vilhelmina J. Characterization of the Radical ScavengingActivity of Lignins-natural Antioxidants[J]. Biores Tech，2004，95：309-317.
69 Shi GF，An LJ，Jiang B，et al. Alpinia protocatechuic acid protects against oxidativedamage in vitro and reduces oxidative stress in vivo[J]. Neurosc letters，2006，403(3)：206-210.
70 Ban J Y，Jeon S Y，Bae K，et al. Catechin and epicatechin from Smilacis chinaerhizome protect cultured rat cortical neurons against amyloid beta protein(25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation[J].Life Sci，2006，79(24)：2251-2259.
71 Williams R J，Spencer J PE，Catherine R E. Flavonoids：Antioxidants or SignallingMolecules [J]. Free Rad Bio Med，2004，36(7)：838-849.
72 Zhao B L. Antioxidant Effects of Green Tea Polyphenols[J]. Chin Sci Bull，2003，48(4)：315-319.
73 Filipa V，Plockova M，Midrkal J S. Resveratrol and its Antioxidant andAntimicrobial Effectiveness[J]. Food Chem，2003，83：585-593.
74钟森，陈文超，徐永强，等.三七总皂苷对脑缺血再灌注损伤大鼠神经干细胞相关调节因子及脑细胞凋亡的影响[J].中国中医急症，2010，19(2)：279-282.
75 Bae E A，Hyun Y J，Choo M K，et al. Protective effect of fermented red ginseng ona transient focal ischemic rats[J]. Arch Pharm Res，2004，27(11)：1136-1140.
76 Haitong Wan，Huiyuan Zhu，Mei，et al. Protective effect of chuanxiongzine puerarinin a rat model of transient middle cerebral artery occlusion-induced focal cerebralischemia[J]. Nuclear Medicine Communications，2008，29(12)：1113-1122.
77蔡青，黄淑芸，谭俊珍，等.丹参酮B钠盐对局灶性脑缺血/再灌注大鼠海马神经元的保护作用[J].辽宁中医药大学学报，2011，13(1)：50-53.
78张春军，郑运马，杨国宏，等.黄芪预处理对大鼠脑缺血再灌注损伤的保护作用研究进展[J].牡丹江医学院学报，2010，31(6)：59-61.
79蒋平，陈鸣，吕军，等.丹参酮ⅡA对阿尔茨海默病模型大鼠海马MMP-2、iNOS表达及自由基释放的影响[J].第二军医大学学报，2010，31(4)：380-384.
80 Changmu Chen，Shing-Hwa Liu，Shoeiyn Linshiau. Honokiol，a neuroprotectantagainst mouse cerebral ischaemia，mediated by preserving Na+，K+-ATPase activityand mitochondrial functions[J]. Basic＆Clinical Pharmacology＆Toxicology，2007，101(2)：108-116.
81王景叶，于榕，姚明辉.红景天苷对缺血再灌注大鼠脑组织损伤的保护作用[J].中华中医药杂志，2010，25(3)：456-459.
82余昌东，邱雄泉，梅全喜.三七总皂苷对脑血管的药理作用研究新进展[J].中国医疗前沿，2008，3(14)：37-39.
83刘慧莲.人参皂甙的药理作用研究进展[J].潍坊学院学报，2009，9(2)：106-108.
84陈勇，孙爱民，陈智贤，等.人参皂甙Rg1对NOS的调控在海马神经元放射性损伤防护中的意义[J].南方医科大学学报，2010，30(7)：1522-1525.
85 He-Song Zeng，Zheng-Xiang Liu，Xiao-Chun Liu. Effects of ginsenoside-Re onanti-cardiomyocyte apoptosis and the expression of related gene proteins in ratsafter acute ischemia-reperfusion[J]. Rehatilitation of TRradltional ChineseMedicine，2004，8(21)：2386-2388 .
86宋晓斌，何波，陈鹏，等.三七皂苷Rg1对动物学习记忆功能的影响及其机理研究[J].昆明医学院学报，2010，31(6)：21-27.
87刘娟，刘颖.丹参药理活性成分研究进展[J].辽宁中医药大学学报，2010，12(7)：15-17.
88卜丽梅，关凤英，乔萍，等.没食子酸对大鼠缺血再灌注损伤后细胞凋亡的保护作用及机制研究[J].中国实验诊断学，2010，14(11)：1693-1697.
89 Huafeng Zhang，Xiamin Hu，Lanxin Wang，et al. Fandian Zeng.Protective effectsof scutellarin against cerebral ischemia in rats：evidence for inhibition of theapoptosis-inducing factor pathway[J]. Planta Medica，2009，75(2)：121-126.
90 Xu X H，Zhang S M，Zhang L，et al. The neuroprotection of puerarin againstcerebral ischemia is associated with the prevention of apoptosis in rats[J]. PlantaMed，2005，71(7)：585-591.
91 Jiang B，Bao Y M，Li Z G，et al. Protection by puerarin against MPP+-inducedneurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction andcaspase-3-like activation[J]. Neurosci Res，2005，53(2)：183-188.
2王耀辉，张路，韩嫣然.脑血管疾病康复护理[J].吉林医学，2011，32(2)：407-408.
3陈跃红，王宝丽.脑血管疾病患者生活质量调查[J].慢性病学杂志，2010，12(10)：1368.
4奥俊青.脑血管疾病的心理护理[J].中国现代药物应用，2011，5(1)：188.
5 Bordet R，Ouk T，Onténiente B，et al. Cerebral ischaemia：tomorrow's therapeutictracks[J]. Med Sci(Paris)，2009，25(10)：847-854.
6王树，薛贵平，张丹参，等.大黄酚对脑缺血再灌注小鼠学习记忆障碍及耐缺氧的影响[J].陕西医学杂志，2008，37(4)：402-404.
7张丹参，张力，薛贵平，等.大黄酚的抗衰老作用[J].中国医院药学杂志，2005，25(1)：15-17.
8王树，张丹参，张力，等.大黄酚对脑缺血再灌注小鼠记忆功能的保护作用[J].中国老年学杂志，2009，29(15)：1934-1936.
9王树，张力，张丹参，等.大黄酚对脑缺血再灌注小鼠探索功能的影响[J].时珍国医国药，2007，18(12)：3011-3013.
10董晓华，张丹参.大黄酚对Aβ25-35所致AD大鼠学习记忆及LTP的影响[J].中国药理学通报，2009，25(5)：682-685.
11刘久青，李茂星.大黄游离蒽醌固体分散体的制备及溶出研究[J].中国医药指南，2010，8(4)：35-37.
12黄红娜，张丹参，郑晓霞，等.大黄酚聚氰基丙烯酸丁酯纳米囊的制备工艺及质量研究[J].中草药，2010，41(4)：547-550.
13 Koffi A A，Agnely F，Ponchel G，et al. Modulation of the rheological andmucoadhesive properties of thermosensitive poloxamer-based hydrogels intendedfor the rectal administration of quinine[J]. European Journal of PharmaceuticalSciences，2006，27(4)：328-335.
14 Mura P，Maestrelli F，Gonzalez-Rodriguez ML，et al. Development，characterization and in vivo evaluation of benzocaine-loaded liposomes[J]. Eur JPharm Biopharm，2007，67(1)：86-95.
15 Himori N，Watanabe H，Akaike N，et al. Cerebral ischemia model with consciousmice：involvement of NMDA receptor activation and derangement of learning andmemory ability[J]. Pharmacol Meth，1990，23：311-327.
16 MeCann SK，DustingG J，Roulston C L. Early increase of Nox4 NADPH oxidaseand super oxide generation following endothelin-1-induced stroke in consciousrats[J]. JNeurosciRes，2008，86(11)：2524-2534.
17 Terao Y，OhtaH，Oda A，et al. Macrophage inflammatory protein-3alpha plays a keyrole in the inflammatory cascade in rat focal cerebral ischemia[J]. Neurosci Res，2009，64(1)：75-82.
18 DaiH B，FuQ L，ShenY，et al. The histamineH3 receptor antagonist clobenpropitenhancesGABA release to protect againstNMDA-induced excitotoxicity throughthe cAMP/protein kinase A pathway in cultured cortical neurons[J].EurJPharmacol，2007，563(1/3)：117-123.
19李春颖，曲娴，李东盈.反复脑缺血再灌注对小鼠学习记忆和脑内氧化损伤的影响[J].北华大学学报，2009，10(4)：322-326.
20 St-Pierre J，Buckinghan JA，Roebuck SJ，et al. Topology of superoxide productionfrom different sites in the mitochondrial electron transport chain[J]. J Biol Chem，2002，277(47)：44784-44790.
21 Devi SA，Kiran TR. Regional responses in antioxidant system to exercise trainingand dietary Vitamin E in aging rat brain[J]. Neurobiol Aging，2004，25：501-508.
22 Grey AD. The reductive hotspot hypothesis of mammalian aging：membranemetabolism magnifies mutant mitochondrial mischief[J]. Eur J Biochem，2002，269(8)：2003.
23王树，张丹参，薛贵平，等.大黄酚对脑缺血再灌注小鼠脑组织H2O2和CAT的影响[J].中药药理与临床，2008，24(4)：22-24.
24 Sandhu SK，Kaur G. Alterations in oxidative stress scavenger system in aging ratbrain and Iumphocytes[J]. Biogerontology.2002，3(3)：161-173.
25 Giardino R，Giavaresi G，Fini M，et al. The role of different ehemical modificationsof superoxide dismutase in preventing a prolonged muscular ischemis/reperfusioninjury[J]. Artif Cells Blood Immubiol Biotechmol.2002，30(3)：189-198.
26况时祥，刘继刚，肖燕，等.脑通胶囊对血管性痴呆大鼠学习记忆能力及脑组织NO、SOD含量的影响[J].中国中医急症，2010，19(8)：1368-1370.
27蔡建伟，吴颢昕，晋光荣，等.双根清脑煎剂对血管性痴呆大鼠模型脑海马组织及血清SOD MDA的影响[J].中华中医药学刊，2007，25(6)：1167-1168.
28杨文明，王时光，洪亮，等.智脑胶囊对血管性痴呆模型大鼠行为学及脑组织SOD活性、MDA含量的影响[J].中西医结合心脑血管病杂志，2007，5(9)：833-835.
29张会珍，李文丽，马小顺，等.电针对拟血管性痴呆小鼠SOD和MDA的影响[J].中国中医基础医学杂志，2010，16(11)：1034-1035.
30顾饶胜，王艳春，刘微，等.大豆异黄酮对急性衰老小鼠学习记忆障碍的改善作用[J].吉林医药学院学报，2007，28(3)：125-127.
31刘秋芳，逯晓波，靳翠红，等.碱性电解水对D-半乳糖致小鼠血液和肝、肾、脑组织脂质过氧化作用的影响[J].环境与健康杂志，2010，27(2)：105-107.
32张晓杰，张静艳，赵坤，等.通络救脑口服液对AD大鼠海马单胺氧化酶活性、脂褐素含量的影响[J].中国老年学杂志，2010，30，(18)：2634-2636.
33薛茜，邹玉安，韩敏，等.康脑液对脑缺血再灌注损伤大鼠血清及脑组织匀浆SOD、MDA、NO的影响[J].中西医结合心脑血管病杂志，2010，8(6)：709-710.
34 Tsai SK，Hung LM，Fu YT，et al. Resveratrol neuroprotective effects during focalcerebral ischemia injury via nitric oxidemechanism in rats[J]. JVasc Surg，2007，46(2)：346-353.
35 Anctil M，Poulain I，Pelletier C. Nitric oxide modulates peristaltic muscle activityassociated with fluid circulation in the sea pansy Renilla koellikeri[J]. JExp Biol，2005，208(10)：2005-2017.
36熊文欣，李文峰.硫化氢对脑缺血再灌注损伤大鼠血清及脑组织NO和NOS的影响[J].现代中西医结合杂志，2010，19(16)：1971-1972.
37袁野，熊缨，杨俊卿，等.单胺氧化酶B在神经元退行性变模型中的意义研究[J].重庆医科大学学报，2006，31(5)：681-683，714.
38姚五湖，龙斌，周日笑.贯叶连翘提取物提高衰老小鼠学习记忆能力的研究[J].今日药学，2010，20(4)：37-39.
39戴恩来，马鸿斌，欧秀梅，等.通络益智散对拟血管性痴呆大鼠AChE，MDA和SOD的影响[J].甘肃中医，2006，19(5)：38-40.
40苗艳艳，苗明三，马霄，等.五味子醇提部位对反复脑缺血再灌注模型小鼠脑能量代谢的影响[J].中华中医药杂志，2009，24(9)：1207-1209.
41杨牧祥，武常生，于文涛，等.醒脑启智胶囊对血管性痴呆小鼠脑组织海马区乙酰胆碱酯酶的影响[J].北京中医药大学学报，2006，29(3)：177-180.
42刘杰，华赟鹏，谭敏谊，等.芍药苷注射液对脑缺血-再灌注沙土鼠脑组织中兴奋性氨基酸及腺苷含量的影响[J].中药材，2010，33(9)：1456-1460.
43张新春，吕光耀，秦秀德.益气活血中药对大鼠缺血-再灌注脑组织中氨基酸含量的影响[J].中国医药导报，2009，6(22)：30-32.
44陆晖，吴云虎，陆艳玲，等.酸枣仁皂苷A对脑缺血再灌注损伤大鼠神经行为学及脑组织氨基酸含量的影响[J].广西中医药，2008，31(6)：47-50.
45颜天华，贾莹，杨伟，等.红景天苷对大鼠局灶性脑缺血/再灌注损伤的保护作用[J].中国药理学通报，2008，24(11)：1521-1524.
46 Liu F，Schafer DP，McCullough LD. TTC，fluoro-Jade B and NeuN stainingconfirm evolving phases of infarction induced by middle cerebral arteryocclusion[J]. J Neurosci Methods，2009，179(1)：1-8.
47王红艳，李威，孙博谦，等.全脑缺血再灌注小鼠额叶、海马区神经元损伤的病理研究[J].中国老年学杂志，2006，26(8)：1088-1090.
48 Yu J，Novgorodov SA，Chudakova D，et al. JNK3 signaling pathway activatesceramide synthase leading to mitochondrial dysfunction[J]. J Biol Chem，2007，3：1-24.
49张兴毅，宋国林，韩江全，等.全脑缺血再灌注后大鼠海马回中Bcl-2及Bax的表达[J].西部医学，2009，21(11)：1850-1852.
50 Yang XF，He W，Lu WH，et al. Effects of seutellarin on liver funetion after brainischemia/reperfusion in rats[J]. Acta Pharmacol Sin，2003，24(11)：1118-1124.
1 Fang Y Z，Yang S，Wu G Y. Free Radicals，Antioxidants，and Nutrition[J].Nutrition，2002，18(10)：872.
2 Cai Y Z，Luo Q，Sun M. Antioxidant Activity and Phenolic compounds of 112Traditional Chinese Medicinal Plants Associated with Anticancer[J]. Life Sci，2004，74：2157-2184.
3 Gerhardt G，Cass W，Yi A，et al. Changes in somatodendritic bur not terminaldopamine regulation in rhesus monkeys[J]. J Neurochem，2002，80(1)：168-177.
4 Siles E.Martinez-Lara E，Canuedo A，et al. Age-related changes of the nitric oxidesystem in the rat brain[J]. Brain Res，2002，956(2)：385-392.
5 Goncharova ND，Lapin BA，Khavinson VKh. Age-associated endocrinedysfunctions and approaches to their correction[J]. Bull Exp Biol Med，2002，134(5)：417-421.
6 Trazona R，Solana R，Ouyang Q，et al. Basic biology and clinical impact ofimmunosenescence[J]. Exp Gerontol 2002，37：183.
7 Tarazona R，Solana R，Ouyang Q，et al. Basic biology and elinical impact ofimmunosenescence[J]. Exp Gerontol，2002，37：183.
8房林，赵振民.皮肤衰老机制的研究进展[J].人民军医，2010，53(2)：149-152.
9 Etzev DE，Lolov SR，Usunoff KG. Aging and synaptic changes in theparaventricular hypothalamic nucleus of the rat[J]. Acta Physiol Pharmacol Bulg，2003，27(2-3)：75-82.
10 Wu G，Yan S. Analysis of distributions of amino acids in the primary structure ofapoptosis regulator Bcl-2 family according to the random mechanism[J]. JBiochem Mol Biol Biophys，2002，6(6)：407-414.
11 Takeuchi A，Mishina Y，Miyaishi O，et al. Heterozygosity with respect to Zfp148causes complete loss of fetal germ cells during mouse embryogenesis[J]. NatGenet，2003，33(2)：172-176.
12杨茂林，孟思进.线粒体氧化损伤在衰老发生机制中的作用[J].医学综述，2010，16(9)：1297-1300.
13张丹丹，白云燕.衰老分子生物学研究进展[J].黑龙江医药科学，2008，31(6)：82-83.
14高凌云，李国栋，童坦君.延缓衰老相关的小分子物质研究进展[J].生物化学与生物物理进展，2010，37(9)：932-938.
15 Brunori M，Luciano P，Gilson E，et al. The telomerase cycle：normol andpathological aspects[J]. J Mol Med，2005，83(4)：244～257.
16彭艳华，王跃.细胞衰老与机体老化关系的研究进展[J].中国老年学杂志，2008，28(9)：932-934.
17 Zhang ZC，Schmitt MT，Mumford JL. Effects of arsenic on telomerase andtelomeres in relation to cell proliferation and apoptosis in human keratinocytes andleukemia cells in vitro[J]. Carcinogenesis，2003，24(11)：1811-1817.
18 Phaneuf S，Leeuwenburgh C. Cytochrome c release from mitochondria in the agingheart：a possible mechanism for apoptosis with age[J]. Am J Physol Regul IntegrComp Physol，2002，282(2)：R423-430.
19古彦铮，强亦忠，倪祥庭，等.干细胞的衰老学说[J].中国组织工程研究与临床康复2008，12(34)：6731-6734.
20彭立伟，来吉祥，何聪芬，等.非酶糖基化与皮肤衰老的研究进展[J].中国老年学杂志，2010，30(20)：3027-3029.
21 Stadtman ER，Levine RL. Free radical-mediated oxidation of free amino acids andamino acid residues in proteins[J]. Amino Acids，2003，25(3-4)：207-218.
22周文丽，张建鹏，冯伟华，等.脑衰老机制与脑疾病的关系[J].生命的化学，2008，28(4)：435-438.
23于海忠，赵淑敏，葛志华. D-半乳糖衰老大鼠下颌下腺超微结构的改变[J].河北医药，2009，31(11)：1287-1288.
24雷秀娟，冯凯，孙立伟，等.人参皂苷抗衰老机制的研究进展[J].氨基酸和生物资源，2010，32(1)：44-47.
25杨吉平，赖红，方欣，等.人参皂苷Rb1对阿尔茨海默病大鼠海马结构β-淀粉样蛋白表达的影响[J].中国组织化学与细胞化学杂志，2008，17(4)：301-305.
26彭书玲，郭兆安.三七总皂苷的作用机制研究进展[J].中国中西医结合急救杂志，2008，15(1)：63-64.
27齐艳萍，李和平，陈雪龙.鹿茸药理作用的研究进展[J].经济动物学报，2008，12(1)：54-55.
28 Hongping Pan，Li Gao. Protecting Mechanism of Puerarin on the Brain Neurocyteof Rat in Acute Local Ischemia Brain Injury and Local CerebralIschemia-reperfusion Injury[J]. Journal of the Pharmaceutical Society of Japan，2008，128(11)：1689-1698.
29 Li Gao，Xunming Ji，Juexian Song，et al. Puerarin protects against ischemic braininjury in a rat model of transient focal ischemia[J]. Neurological Research，2009，31(4)：402-406.
30 Xingsan Tang，Yazhen Ma. Protective effects of Breviscapine injection against focalcerebral ischemia-reperfusion injury in rats[J]. Chinese Journal of ClinicalRehabilitation，2005，9(29)：243-245.
31钟振国，吴登攀，王进声，等.三七总皂苷对老年性痴呆动物模型快速老化小鼠大脑Aβ1-40、Aβ1-42表达的影响[J].中药材，2009，32(1)：82-85.
32 Tombaugh GC，Rowe WB，Chow AR，et al. Theta-frequency syna-pticpotentiationin CA1 invito distinguishes cognitively impaired from unimpaired aged Fischer
344 rats[J]. Neuro-sci，2002，22(22)：9932-9940.
33刘霞，包翠芬，魏嘉，等.人参皂苷Rg1对脑缺血-再灌注大鼠海马CA1区神经元的保护作用及机制[J].解剖科学进展，2010，16(2)：177-180.
34陈超杰，张文生.七治疗阿尔茨海默氏病的研究进展[J].时珍国医国药，2008，19(7)：1559-1564.
35李维祖，李卫平，尹艳艳，等.黄芪总苷及黄芪甲苷对糖皮质激素诱导老前期小鼠记忆障碍的保护作用及其机制[J].安徽医药，2008，12(7)：592-594.
36 Chan RYK，ChenWF，DongA，et al. Estrogen-like activity of Ginsenoside Rg1derived fromPanax notoginseng[J]. J Clin Endocrinol Metab，2006，87(8)：3691-3695.
37 Byung SK，Tae SK，Cheorl HK. Salviae miltiorrhizae radix inhibits superoxidegeneration by activated rat microglias and mimics the action of amphetamine on invitro rat striatal dopamine release[J]. Neurochem Res，2004，29(10)：1837-1845.
38钟桂书，何渊民，廖勇梅.雷公藤、人参与芳维甲酸乙酯对培养光老化真皮成纤维细胞的影响[J].中国组织工程研究与临床康复，2010，14(2)：267-271.
39周燕，宁宗，田磊，等.三七总皂苷对大鼠海马CA1区兴奋性突触活动的影响[J].广西医科大学学报，2010，27(4)：532-535.
40 Sparg S G，Light M E，Staden J V. Biological Activities and Distribution of PlantSaponins[J]. J Ethnopharm，2004，94：219-243.
41栗平.何首乌抗衰药理与临床应用[J].慢性病学杂志，2010，12(10)：1271-1272.
42李娜，贾芳.人参皂苷治疗阿尔茨海默病作用机制的研究进展[J].医学综述，2008，14(17)：2691-2693.
43 Tian J，Fu F，Geng M，et al. Neuroprotective effect of 20(S)-ginsenoside Rg3 oncerebral ischemia in rats[J]. Neurosci Lett，2005，374(2)：92-97.
44 Jeong S M，Lee J H，Kim J H，et al. Stereospecificity of ginsenoside Rg3 action onion channels[J]. Mol Cells，2004，18(3)：245-251.
45 Hua Li，Changqing Deng，Beiyang Chen，et al. Total saponins of Panax notoginsengmodulate the expression of caspases and attenuate apoptosis in rats following focalcerebral ischemia-reperfusion [J]. Journal of Ethnopharmacology，2009，121(3)：412-418.
46王东吉，尚改萍，武凡，等.三七皂苷单体Rg1对大鼠脑缺血再灌注损伤保护作用的研究[J].中国中医基础医学杂志，2008，14(7)：513-515.
47刘婕，许浚，张铁军.苯丙素苷类化合物防治神经退行性疾病的研究进展[J].药物评价研究，2009，32(1)：62-66.
48 Zhanjun Zhang，Runguo Wu，Pengtao Li，et al. Baicalin administration is effectivein positive regulation of twenty-four ischemia/reperfusion-related proteinsidentified by a proteomic study[J]. Neurochemistry International，2009，54(8)：488-496.
49张凤普.人参皂苷的作用机理研究[J].临床合理用药，2010，3(21)：158.
50 Ji XY，Tan Benny Kg，Zhu YC，et al. Comparison of cardioprotective effects usingramipril and DanShen for the treatment of acute myocardial infarction in rats[J].Life Sci，2003，73：1413-1426.
51 Ikeda K，Negishi H，Yamori Y. Antioxidant nutrients and hypoxia/ischemia braininjury in rodents[J]. Toxicology，2003，189(1-2)：55-61.
52 Chandrasekaran K，Mehrabian Z，Spinnewyn B，et al. Bilobailide，a componentof theGinkgo bilobaextract (EGB761)，protects against neural death in global brainischemia and in glutamate-induced excitotoxicity[J]. Cell Mol Biol，2002，48(6)：663-669.
53 Giuseppe G，Peter J O. Potential Toxicity of Flavonoids and other DietaryPhenolics：Significance for their Chemopreventive and Anticancer Properties[J].Free Rad Bio Med，2004，37(3)：287-303.
54 Clarkson AN，Liu H，Pearson L，et al. Neuroprotective effects of sperminefollowing hypoxia-ischemia-induced braidamage：a mechanistic study[J]. FASEBJ，2004，18(10)：1114-1116.
55 Wei I H，Wu Y C，Wen C Y，et al. Green tea polypheno(-)-epigallocatechin gallateattenuates the neuronal NADPH d/nNOS expression in the nodose ganglion ofhypoxic rat[J]. Brain Res，2004，999(1)：73-80.
56谢甦，李丽红，李丽.三七总皂苷抗衰老的实验研究[J].世界中西医结合杂志，2008，3(2)：86-88.
57 Wen J X，Mo Y，Tai F F，et al. Partial neuroprotective effect of pretreatment withtanshinone IIA on neonatal hypoxiaischemia brain damage[J]. Pediatr Res，2005，58(4)：784-790.
58薛亮，范英昌，李庆雯，等.丹酚酸B对离体培养内皮祖细胞VEGF、bFGFmRNA表达及抗氧化酶活性的影响[J].中国老年学杂志，2011，31(2)：236-238.
59祝飞虹，高根德.中药治疗老年性痴呆临床研究近况[J].实用中医药杂志，2011，27(1)：68-69.
60杜娟，陈虹.肉苁蓉及其有效成分对脑损伤的保护作用研究进展[J].时珍国医国药，2010，21(9)：2333-2334.
61 Toyokuni S，Tanaka T，Kawaguchi N. R，et al. Effects of the phenolic contents ofmauritianendemic plantextracts on promoter activities of antioxidant enzymes[J].Free Radic Res. 2003，37：1215.
62 Hyang D G，Kwang H L，Hyoung J K，et al. Neuroprotective effects ofantioxidative flavonoids，quercetin(+)-dihydroquercetin and quercetin 3-methylether isolated from Opuntia ficus-indica Var saboten[J]. Brain Res，2003，965(1-2)：130-136.
63 Choi E J，Lee BH，Chee KM. Long-term combined administration of quercetin anddaidzein inhibits quercetin-induced suppression of glutathione antioxidantdefenses[J]. Food ChemToxicol，2005，43(5)：793-798.
64 GronebergD.A，Grosse-Siestrup C and FischerA. In vitromodels to studyhepatotoxicity[J]. ToxicolPalhol，2002，30：394.
65 Zbarsky V，Datla K P，Parkar S，et al. Neuroprotective properties of the naturalphenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a
6-OHDA model of Parkinson’s disease[J]. Free Radic Res，2005，39(10)：1119-1125.
66 Luo Y，Qin Z，Hong Z，et al. Astragaloside IV protects against ischemic brain injuryin a murine model of transient focal ischemia[J]. Neurosci Lett，2004，363(3)：218-223.
67 Wei X B，Liu H Q，Sun X，et al. Hydroxysafflor yellow A protects rat brains againstischemia-reperfusion injury by antioxidant action[J]. Neurosci Lett，2005，386(1)：58-62.
68 Tatiana D，Galina T，Vilhelmina J. Characterization of the Radical ScavengingActivity of Lignins-natural Antioxidants[J]. Biores Tech，2004，95：309-317.
69 Shi GF，An LJ，Jiang B，et al. Alpinia protocatechuic acid protects against oxidativedamage in vitro and reduces oxidative stress in vivo[J]. Neurosc letters，2006，403(3)：206-210.
70 Ban J Y，Jeon S Y，Bae K，et al. Catechin and epicatechin from Smilacis chinaerhizome protect cultured rat cortical neurons against amyloid beta protein(25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation[J].Life Sci，2006，79(24)：2251-2259.
71 Williams R J，Spencer J PE，Catherine R E. Flavonoids：Antioxidants or SignallingMolecules [J]. Free Rad Bio Med，2004，36(7)：838-849.
72 Zhao B L. Antioxidant Effects of Green Tea Polyphenols[J]. Chin Sci Bull，2003，48(4)：315-319.
73 Filipa V，Plockova M，Midrkal J S. Resveratrol and its Antioxidant andAntimicrobial Effectiveness[J]. Food Chem，2003，83：585-593.
74钟森，陈文超，徐永强，等.三七总皂苷对脑缺血再灌注损伤大鼠神经干细胞相关调节因子及脑细胞凋亡的影响[J].中国中医急症，2010，19(2)：279-282.
75 Bae E A，Hyun Y J，Choo M K，et al. Protective effect of fermented red ginseng ona transient focal ischemic rats[J]. Arch Pharm Res，2004，27(11)：1136-1140.
76 Haitong Wan，Huiyuan Zhu，Mei，et al. Protective effect of chuanxiongzine puerarinin a rat model of transient middle cerebral artery occlusion-induced focal cerebralischemia[J]. Nuclear Medicine Communications，2008，29(12)：1113-1122.
77蔡青，黄淑芸，谭俊珍，等.丹参酮B钠盐对局灶性脑缺血/再灌注大鼠海马神经元的保护作用[J].辽宁中医药大学学报，2011，13(1)：50-53.
78张春军，郑运马，杨国宏，等.黄芪预处理对大鼠脑缺血再灌注损伤的保护作用研究进展[J].牡丹江医学院学报，2010，31(6)：59-61.
79蒋平，陈鸣，吕军，等.丹参酮ⅡA对阿尔茨海默病模型大鼠海马MMP-2、iNOS表达及自由基释放的影响[J].第二军医大学学报，2010，31(4)：380-384.
80 Changmu Chen，Shing-Hwa Liu，Shoeiyn Linshiau. Honokiol，a neuroprotectantagainst mouse cerebral ischaemia，mediated by preserving Na+，K+-ATPase activityand mitochondrial functions[J]. Basic＆Clinical Pharmacology＆Toxicology，2007，101(2)：108-116.
81王景叶，于榕，姚明辉.红景天苷对缺血再灌注大鼠脑组织损伤的保护作用[J].中华中医药杂志，2010，25(3)：456-459.
82余昌东，邱雄泉，梅全喜.三七总皂苷对脑血管的药理作用研究新进展[J].中国医疗前沿，2008，3(14)：37-39.
83刘慧莲.人参皂甙的药理作用研究进展[J].潍坊学院学报，2009，9(2)：106-108.
84陈勇，孙爱民，陈智贤，等.人参皂甙Rg1对NOS的调控在海马神经元放射性损伤防护中的意义[J].南方医科大学学报，2010，30(7)：1522-1525.
85 He-Song Zeng，Zheng-Xiang Liu，Xiao-Chun Liu. Effects of ginsenoside-Re onanti-cardiomyocyte apoptosis and the expression of related gene proteins in ratsafter acute ischemia-reperfusion[J]. Rehatilitation of TRradltional ChineseMedicine，2004，8(21)：2386-2388 .
86宋晓斌，何波，陈鹏，等.三七皂苷Rg1对动物学习记忆功能的影响及其机理研究[J].昆明医学院学报，2010，31(6)：21-27.
87刘娟，刘颖.丹参药理活性成分研究进展[J].辽宁中医药大学学报，2010，12(7)：15-17.
88卜丽梅，关凤英，乔萍，等.没食子酸对大鼠缺血再灌注损伤后细胞凋亡的保护作用及机制研究[J].中国实验诊断学，2010，14(11)：1693-1697.
89 Huafeng Zhang，Xiamin Hu，Lanxin Wang，et al. Fandian Zeng.Protective effectsof scutellarin against cerebral ischemia in rats：evidence for inhibition of theapoptosis-inducing factor pathway[J]. Planta Medica，2009，75(2)：121-126.
90 Xu X H，Zhang S M，Zhang L，et al. The neuroprotection of puerarin againstcerebral ischemia is associated with the prevention of apoptosis in rats[J]. PlantaMed，2005，71(7)：585-591.
91 Jiang B，Bao Y M，Li Z G，et al. Protection by puerarin against MPP+-inducedneurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction andcaspase-3-like activation[J]. Neurosci Res，2005，53(2)：183-188.