当归芍药散抗衰老活性的药效学和药代动力学研究
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摘要
阿尔茨海默病(Alzheimer's Disease, AD),又称老年性痴呆病[1],是一种进行性认知障碍和记忆力损害为主的中枢神经系统退行性疾病,出现的全面持久的智能减退,包括记忆力、判断力、计算力、思维能力和语言功能的减退,丧失独立工作能力和生活能力。其病理变化主要表现为脑内胆碱能神经元的原发性变性和减少、β样淀粉样蛋白沉积脑内所形成的老年斑以及神经原纤维缠结。
随着人类寿命的延长,全球进入老龄化社会。目前AD已成为继癌症、心血管疾病和中风之后威胁老年人健康的又一大疾病。我国的流行病学调查资料显示,AD在65岁以上人群中的发病率为3%-5%,接近西方发达国家水平。衰老、基因突变及遗传为AD明确的致病原因,此外炎症及应激,神经递质减少,代谢异常等都为其可能的致病原因[2-3]。
AD至今无有效的治疗方法,目前主要是对症治疗。多年以来,乙酰胆碱酯酶抑制剂如加兰他敏、石杉碱甲,谷氨酸受体调控剂如盐酸美金刚胺,脑循环改善剂如尼麦角林等,γ-氨基丁酸类促智药如吡拉西坦等被广泛应用于AD的治疗[4-7]。但由于AD病人需要长期服药,西药的副作用比较大,不断寻求副作用小的药物以有效地治疗AD成为现代社会人们广泛关注的重要问题。脏腑失调,气血亏虚,脑髓失养是形成AD的必要条件,肾之精气化精生髓汇聚而为脑,五脏气血之精华皆上荣于脑,脑髓之功能发挥,须依赖气血滋养。老年人脏腑功能失调,气血生化乏源,脑髓得不到充足的气血滋养,久则神明受损,功能低下。中药当归芍药散具有整体作用的特点,与临床常用的抗老年性痴呆化学药物相比,它们的副作用比较小,不易产生耐药性,有明显前景和优势[8-10]。当归芍药散(DangguiShaoyao San,以下简称DSS)是《金匮要略》所载著名方剂[1],由当归、芍药、川芎、茯苓、白术、泽泻(1：5.3：2.6：1.3：1.3：2.6)六味药组成。当归具有补血活血,调经止痛,润肠通便的功效。芍药有养血敛阴,补而不腻,柔肝缓中,止痛收汗功效。当归芍药散具有养血调肝,健脾利湿,养血益脾等功效。原方主治“妇人妊娠诸疾痛”以及“妇人杂病腹中诸疾痛”。目前有研究将DSS转变为治疗老年性痴呆的有效方剂,疗效确切[12-14]。前期的研究基础：本课题在国家自然基金“当归芍药散化学成分、剂量配比与药效学关系”的研究项目资助的支持下,采用现代药理研究方法,研究当归芍药散的抗痴呆作用。采用D-半乳糖致致小鼠亚急性衰老的经典动物模型,考察了当归芍药散及单味药材提取液对动物神经系统的影响[15-17],结果发现当归芍药散能显著改善学习记忆能力。中药方剂是中医临床治疗疾病的主要手段,药物的合理配伍可以相使相须,达到一种优化组合的整体疗效。当归芍药散的抗老年痴呆作用与其组方药材有着密切的关系,又非这些药材中化学成分的简单加和。基于以上原因,研究设计将中药化学、质量控制以及药理学技术紧密结合,以确定当归芍药散抗老年痴呆作用的物质基础。
目的
在抗老年痴呆药理活性指导下,对当归芍药散(DSS)(当归、芍药、川芎、茯苓、白术、泽泻(1：5.3：2.6：1.3：1.3：2.6))药效物质的提取工艺、化学成分及其含量分析、指纹图谱、化学成分在动物体内代谢等进行了相关研究,旨在为当归芍药散的二次开发提供科学依据。
方法
采用药理学实验研究方法,对当归芍药散水提和醇提部位进行了抗老年痴呆的药理筛选研究。建立D-半乳糖联合AlCl3所致亚急性衰老小鼠的模型,考察各组小鼠体重变化,统计各组小鼠八臂迷宫的工作记忆错误次数、参考记忆错误次数、错误总数次数。
采用Lg(34)正交表对抗痴呆药效最好的当归芍药散醇提部位(以下简称当归芍药散)的提取工艺进行了研究。因素选择：乙醇浓度(A)、提取次数(B)、提取时间(C)、乙醇用量(D),每个因素设置3个水平。选择多指标成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行综合测评,确定各因素的最佳提取工艺水平。在此基础上,采用系统溶剂分离法,对当归芍药散(DSS)进行了部位分离,分离了石油醚部位(DSS-P)、乙酸乙酯部位(DSS-E)、正丁醇部位(DSS-N)、水部位(DSS-W),以进行化学成分和药效学研究。
建立了当归芍药散(DSS)及4个部位的主要活性成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行了HPLC-DAD含量分析方法。色谱条件为：色谱柱syncronisC18(4.6mm×250mm,5μm)不锈钢柱；流动相：A：乙腈,B：0.1%磷酸,梯度程序条件洗脱；流速：1.Oml/min;柱温：30C°；进样体积：10μL；用二极管检测器进行190nm-400nm全波长扫描,经比较后发现220nm、270nm、320nm处基线平稳,出峰多,峰强度较好,故确定检测波长为220nm、270nm、320nm。λ1=220nm(检测芍药苷),λ2=270nm(检测川芎嗪、白术内酯Ⅰ),λ3=320nm(检测藁本内酯、阿魏酸。对当归芍药散进行了指纹图谱的研究,标定了共有峰,采用中药色谱指纹图谱相似度评价系统进行相似度分析,建立当归芍药散的三波长融合指纹图谱,使用化学对照品和药材对色谱峰进行定性、归属。
药效试验采用体内结合体外研究方法,对当归芍药散(DSS)及石油醚部位(DSS-P)、乙酸乙酯部位(DSS-E)、正丁醇部位(DSS-N)、水部位(DSS-W)进行了抗痴呆筛选研究。(1)利用D-半乳糖联合AlCl3造成小鼠老年痴呆病理的复合模型,出现学习记忆力减退；海马和皮质老年斑形成,促进Aβ的沉积等类似AD病变,造模机制比较接近AD。用八臂迷宫研究动物的行为学变化,检测其生化指标(SOD、MDA、GSH-Px、脂褐素),HE染色观察脑组织病理形态学等方面对当归芍药散及四个部位进行了研究；(2)采用体外清除1,1-二苯基苦基苯肼(DPPH)法对当归芍药散(DSS)及其部位进行抗氧化研究。当有自由基清除齐(?)DPPH存在时,由于与DPPH的单电子配对而使其吸收逐渐消失,其褪色程度与配对电子数成化学计量关系,因而可用分光光度法进行定量分析；(3)选用与多巴胺能神经元有许多相似特征的PC12细胞为实验对象,以过氧化氢(H2O2)诱导PC12细胞损伤,从而建立体外多巴胺能细胞的AD氧化应激模型,研究当归芍药散(DSS)及部位的保护作用,包括：H2O2作用时间和浓度的选择、当归芍药散(DSS)对细胞毒性考察和浓度的选择、当归芍药散(DSS)对细胞存活率的影响。用倒置显微镜观察培养的PC12细胞的形态,用形态学方法证明PC12细胞培养成功。采用150、200、250、300、350、400、450μg/ml浓度的当归芍药散(DSS)分别与PC12细胞共同孵育24h,以MTT法测定细胞存活率。考察当归芍药散对过氧化氢(H202)诱导PC12细胞活性的影响,不同剂量当归芍药散(100、150、200、250、300、350、400μg/ml预孵育PC12细胞2h,再和H2O2共培养6h,以MTT法测定细胞存活率；采用剂量相当于当归芍药散(DSS)(300μg/ml)的各个部位预孵育PC,2细胞2h,再和H2O2共培养6h,以MTT法测定细胞存活率。(4)当归芍药散(DGS)的化学成分与药效相关性研究,利用本章药效实验数据与第二章质量控制的HPLC实验数据,将当归芍药散、乙酸乙酯、正丁醇、水部位得到的化学成分对应的色谱图中各色谱峰Xi与本章药效实验数据(变量Y(Y1：小鼠42天体重增长鼠；Y2：八臂迷宫RME错误次数；Y3：八臂迷宫WME错误次数； Y4：八臂迷宫错误总次数；Y5：八臂迷宫时间；Y6：小鼠血浆SOD含量；Y7：小鼠血浆MDA含量；Y8：小鼠血浆GSH-Px含量；Y9：小鼠脑内脂褐素的含量；Y10：对DPPH清除的影响；Y11：对PC12的存活率))进行相关性统计分析,研究当归芍药散化学成分与药效之间的关系。
基于LC/MS/MS技术分析当归芍药散在大鼠血液和组织中指标性成分,观察其代谢的化学成分谱变化。(1)建立的多成分LC/MS/MS法分析方法学认证的基础上,对单次口服当归芍药散(18g(生药)/Kg)后的大鼠血浆中多种成分的药代动力学进行了初步的研究,考察成分的药代动力学参数。色谱及质谱条件色谱柱为Eclipse XDB C18柱(3.5μm,2.0mm×150mm),流动相为甲醇：10mmol·L-1乙酸铵(90：10),流速为200μL·min-1,样品进样量为20μL,柱温：35℃,分析时间为3.0min,用外标法进行定量分析。采用辅助气化电喷雾离子源(ESI)正离子和负离子分时段监测模式(MRM)进行定量分析,0-2.8min时段负离子监测离子对为：m/z478.9→448.9(芍药苷)和m/z192.8→177.8(阿魏酸),2.8-6min时段正离子监测离子对为：m/z231.4→185.1(白术内酯-Ⅰ)。离子源的雾化气和气帘气均为氮气,流量分别为70、30mL·min-1、碰撞气(氮气)流量为12L·min-1,离子源喷雾电压：-4500V/+4500V,雾化温度：450℃。(2)单次口服当归芍药散(18g(生药)/Kg)后,建立了大鼠的组织样品中当归芍药散多成分的LC/MS/MS分析方法,灌胃当归芍药散给大鼠后,参考芍药苷和阿魏酸血药浓度—时间曲线,确定采样时间点为15min,35min和6h。SD大鼠15只,每组5只,随机分为3组。灌胃当归芍药散后分别于给药后的15min,35min和6h,将大鼠迅速颈椎脱臼处死,分离胃,肠,肺,肾,脑,心,肝,脾等组织(胃,肠组织分别用蒸馏水将内容物冲洗于净,用干燥滤纸吸干),共8种组织。分别进行LC/MS/MS定量分析。阐明当归芍药散多成分在各组织中的分布特点及组织中药物浓度随时间的变化规律。
统计数据均用SPSS13.0统计分析软件处理,显著性水平取α=0.05(双侧),P<0.05为差异有统计学意义。
结果
1.提取工艺的研究
在当归芍药散对D-半乳糖联合AlCl3所致亚急性衰老小鼠的影响实验中发现：42d检测行为学实验结果：与模型组比及当归芍药散醇提部位组(高、低剂量)和水提部位组(高、低剂量)均使小鼠在走完八臂迷宫所用时间、小鼠工作记忆错误、参考记忆错误、错误总数减少,有显著性差异(P<0.05)。各给药组间比较,当归芍药散醇提部位组高剂量(40g/Kg)(以下简称当归芍药散)表现最好。
在确认改善AD小鼠学习能力效果最好的是DSS乙醇浸膏高剂量的药效活性指导下,采用(L34)正交设计实验对当归芍药散的乙醇提取工艺进行了研究,选择多指标芍药苷、阿魏酸、白术内酯、川芎嗪、藁本内酯为定量指标进行综合测评。依据方差分析结果：A、B、D因素均对实验结果差异有显著性(P<0.05),A2优于A1和A3；B2优于B1和B3；D2优于D1和D3,各因素影响程度依次为A>B>D>C,再结合直观分析结果和实际情况综合考虑,确定各因素的最佳水平组合为A2B2C3D2,采用70%乙醇,提取2次,提取3小时,乙醇用量8倍。
2.当归芍药散质量控制的研究
采用HPLC-DAD法分别对当归芍药散(DSS)及石油醚部位(DSS-P)、乙酸乙酯部位(DSS-E)、正丁醇部位(DSS-N)、水部位(DSS-W)的主要活性成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行了含量分析研究,芍药苷lgy=0.97381gx+5.968,r=0.9996,含量在3.46-34.6μg范围内线性关系良好；阿魏酸1gy=1.01971gx+6.728,r=0.9999,含量在0.174-1.74μg1范围内线性关系良好；川芎嗪1gy=0.99731gx+6.105,r=0.9994,含量在5.1-51.0u g范围内线性关系良好；白术内酯Ⅰ1gy=0.16261gx+6.687,r=0.9990,含量在0.952-9.52μg范围内线性关系良好；藁本内酯1gy=0.98161gx+5.161,r=0.9992,含量在8.24-82.4μg范围内线性关系良好。其中当归芍药散中5种成分测定结果分别为：芍药苷5.945mg/g、阿魏酸0.042mg/g、川芎嗪3.930mg/g、白术内酯Ⅰ0.7200mg/g、藁本内酯1.470mg/g；石油醚部位中白术内酯10.1790mg/g、藁本内酯0.8770mg/g；乙酸乙酯部位中芍药苷1.261mg/g、阿魏酸0.037mg/g、川芎嗪3.045mg/g；正丁醇部位中芍药苷2.801mg/g、阿魏酸0.0040mg/g、川芎嗪0.9940mg/g；水部位中芍药苷0.2080mg/g、川芎嗪2.497mg/g。5种成分的含量特点：芍药苷：DSS>DSS-N>DSS-E>DSS-W:阿魏酸：DSS>DSS-E>DSS-N:川芎嗪：DSS>DSS-E>DSS-W>DSS-N>DSS-P:白术内酯Ⅰ：DSS>DSS-P:藁本内酯：DSS>DSS-P。
建立了该当归芍药散的三波长(220nm,270nm,320nm)融合指纹图谱,10批样品230nm指纹图谱有30个共有峰,270nm处有25个共有峰,320nm处有20个共有峰。三波长融合指纹图谱共有32个共有峰,在峰数及含量上兼顾三个特征波长指纹的特点。标定5个对照品芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯的色谱峰,标定了共有峰32个,方法分离度好,重现性和稳定性良好。其中0-68min为220nm波长下的指纹图谱、270nm波长下的指纹图谱,68-90min为320nm波长下的指纹图谱。3.药效试验采用体内结合体外研究方法,对当归芍药散及石油醚部位、乙酸乙酯部位、正丁醇部位、水部位进行了抗衰老筛选研究。
(1)第7d各组小鼠体质量增长数有明显统计学差异(P<0.05)。第14d与阳性组比模型组的质量增长数显著性增加(P<0.01),第21d时,与空白组比较,模型组体质量增长数减少(P<0.01)；与模型组比较,当归芍药散及各部位组体质量增长数增加(P<0.05);42d时与空白组比,模型组体质量增长数减少,有明显统计学差异(P<0.01),与模型组比较,当归芍药散组及各部位组体质量增长数增加(P<0.01或P<0.05)。行为学实验结果：模型组小鼠走完八臂迷宫所用时间、小鼠工作记忆错误次数、参考记忆错误次数、错误总次数高于空白组(P<0.01),造模成功；当归芍药散组及石油醚部位、乙酸乙酯部位、正丁醇部位组小鼠走完八臂迷宫所用时间、工作记忆错误、参考记忆错误、错误总数少于模型组(P<0.01),各给药组间比较,各部位组中乙酸乙酯组和正丁醇组为最好。生化指标(SOD、MDA、GSH-Px、脂褐素)检测结果表明：与空白组比模型组SOD活性明显降低(P<0.01)。与模型组比各个部位明显升高损伤的小鼠血中SOD活性,尤其以当归芍药散和乙酸乙酯部位最好。与空白组比模型组MDA活性明显升高(P<0.01)。与模型组比各个部位明显升高损伤的小鼠血中MDA,差异均具有显著性意义(P<0.01)。尤其以当归芍药散和乙酸乙酯部位最好。与空白组比模型组GSH-Px活性明显降低(P<0.01)。与模型组比各个部位明显升高损伤的小鼠血中GSH-Px活性,差异均具有非常显著性意义(P<0.01)。尤其以当归芍药散、乙酸乙酯部位、正丁醇部位最好。脑内脂褐素结果表明：与空白组比模型组脂褐素明显升高(P<0.01)；与模型组比当归芍药散及各部位明显降低小鼠脑中脂褐素的含量。HE染色的一般形态学光镜下观察：①空白组小鼠皮质区神经细胞结构正常,数量较多,胞核清晰,胞浆丰富,间质无水肿表现,胶质细胞散布于神经细胞之间；海马区锥体细胞层排列紧密,细胞核圆而大,核仁清晰。②模型组皮质区神经细胞减少和胶质细胞增多、细胞排列稀疏、杂乱、有的细胞核固缩、细胞线不清晰,核仁消失,胞浆深染；海马区锥体细胞层数减少,排列稀疏不规则,细胞体积变小,可见核固缩现象。③与模型组比当归芍药散组、乙酸乙酯组、正丁醇组比有明显改善,小鼠皮质区细胞结构排列较整齐、紧密、细胞核可见；皮质区仍有不同程度的神经细胞变性.但变性神经细胞数量明显减少,可见少部分损伤细胞,细胞形态趋于正常,海马区锥体细胞形态、排列基本正常。
(2)由当归芍药散及各部位对DPPH自由基的清除率作曲线可知,当归芍药散、乙酸乙酯、正丁醇、水部位提取物的IC50值分别为0.3518,1.012、1.133、1.371mg/mL,石油醚无IC50值。清除DPPH自由基能力的大小顺序依次为：当归芍药散>乙酸之酯部位>正丁醇部位>水部位>石油醚部位。但是溶液质量浓度在2mg/mL以上时当归芍药散与乙酸乙酯部位清除率接近。
(3)采用不同浓度的当归芍药散的结果发现：与正常细胞比当归芍药散在400、450μg/ml时对PC12细胞明显毒性作用(P<0.05)。不同浓度当归芍药散可拮抗H2O2的作用,细胞存活率明显升高,各剂量下细胞存活率分别为120%、128%、129%、126%、100%、59%、51%,具有明显的剂量相关性(P<0.001),与正常对照组比较差异均具有统计学意义(P<0.05)。采用剂量相当于当归芍药散(300μg/m1)的各部位均可拮抗H,O,的作用,细胞存活率明显升高,与模型组比较差异均具有统计学意义(P<0.05),但是各部位之间无明显差异,有可能当归芍药散在全方整体角度表现出对神经细胞的保护作用。(4)当归芍药散的化学成分与药效相关性研究发现Pearson分析和灰色关联度分析表明：前面所述5个已知指标性成分芍药苷,阿魏酸,川芎嗪,藁本内酯,白术内酯Ⅰ与药效具有高度相关性。
4.当归芍药散大鼠体内多成分的药代动力学研究本实验采用LC/MS/MS法测定各时间点血浆中芍药苷、阿魏酸的含量,灵敏度高、快速、简单、准确、分析时间短,在5分钟内出峰完毕,重现性好,可用于大鼠血浆中芍药苷、阿魏酸的定量,其它成分未能检出,可能是因为该测定方法灵敏度不够高,或可能是这些成分在大鼠体内转化成其他物质有待今后进一步研究。大鼠单次灌胃当归芍药散后,芍药苷的吸收和消除十分迅速,在给药后的血浆中能很快测出较高浓度的芍药苷(大鼠的Cmax出现在前0.5h),药-时曲线呈现双峰现象,约在8-10h出现第2个峰值(浓度低于第1个峰值)。表明该成分体内吸收较快,药物在体内消除较快,半衰期为5.0±1.4h。
大鼠灌胃当归芍药散后,在大鼠体内芍药苷和阿魏酸各组织分布高低顺序如下
(1)大鼠体内15min时各组织中芍药苷浓度顺序为：胃>肝>肺>肠>心>血>肾>>脑：
(2)大鼠体内35min时各组织中芍药苷浓度顺序为：胃>血>肺>肠>肾>肝>心>>脑：
(3)大鼠体内6h时各组织中芍药苷浓度顺序为：胃>血>肠>肾>肝≈肺≈心>脑。
(4)大鼠体内15min时各组织中阿魏酸浓度顺序为：胃>肠>肝>肺>心>血>肾≈脑：
(5)大鼠体内35min时各组织中阿魏酸浓度顺序为：胃>肠>肺>>脑>肝>肾>血>心：
(6)大鼠体内6h时各组织中阿魏酸浓度顺序为：胃>肠>肺>肾>血。
大鼠灌胃当归芍药散后,当归芍药散可迅速分布于全身大多数组织脏器,大鼠5min时各组织芍药苷和阿魏酸浓度达到峰值,这与血药浓度-时间曲线变化一致。6h时大鼠各组织中芍药苷和阿魏酸浓度降至不足峰值时的10%。当归芍药散在大鼠中芍药苷和阿魏酸均以胃肠道中含量最高,脑中含量最低,相对芍药苷来说阿魏酸的组织消除速率比较慢。
结论
研究综合采用化学指纹图谱、药效学、药代动力学等联合研究的模式证明了当归芍药散抗衰老的作用机制。
化学指纹图谱：采用HPLC-DAD法分别对当归芍药散及石油醚部位、乙酸乙酯部位、正丁醇部位、水部位的主要指标性成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行了含量分析研究。发现当归芍药散及各部位中各种成分的含量各有高低。建立了该当归芍药散的三波长(220nm,270nm,320nm)融合指纹图谱,标定5个对照品芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯的色谱峰,标定了共有峰32个,方法分离度好,重现性和稳定性良好,为药效研究提供质量保证。
药效学实验：证明当归芍药散能明显改善D-半乳糖联合Alcl3所致亚急性衰老小鼠行为学的异常和空间认知障碍,提高学习记忆能力；减少自由基对脑组织的氧化损害;改善病理因素造成的实验动物脑内组织形态的异常。体外抗氧化实验证实其抗氧化能力的大小顺序依次为：当归芍药散>乙酸乙酯部位>正丁醇部位>水部位>石油醚部位。体外PC12细胞实验证明当归芍药散和各部位可拮抗H2O2的作用,细胞存活率明显升高,但是各部位之间无明显差异,有可能当归芍药散在整体角度表现出对神经细胞的保护作用。
药代动力学研究表明：大鼠体内血浆芍药苷阿魏酸等是当归芍药散抗痴呆活性的主要有效组分。给大鼠灌胃当归芍药散后,当归芍药散可迅速分布于全身大多数组织脏器,大鼠5min时各组织芍药苷和阿魏酸浓度达到峰值,当归芍药散在大鼠中芍药苷和阿魏酸均以胃肠道中含量最高,脑中含量最低,相对芍药苷来说阿魏酸的组织消除速率比较慢。
当归芍药散的化学成分与药效相关性研究发现Pearson分析和灰色关联度分析表明：前面所述5个已知指标性成分芍药苷,阿魏酸,川芎嗪,藁本内酯,白术内酯Ⅰ与药效具有高度相关性,结合药代动力学结果在实验角度阐明这5个指标性成分作为药效物质基础的科学性。
Alzheimer's disease (AD) is a kind of neuron degenerative disease that causes behavior deterioration and progressive cognitive. Although the causes of AD diverse are complex, but the histopathoiogical features are almost exactly the same, such as the formation of nerve cells outside the A β amyloidal protein, which was seen as a direct result in the pathological causes of AD, the formation of neuron fibril tangles in nerve cells, the feature of senile plaques, decrease in the number of nerve cells.
As the life of human being increase, AD often can find in the elderly over65more and more. The number of people with AD has increased too, which is heavy burden to AD patient's families and our society.
Currently there is no certain doctrine about etiology and pathogenesis of AD, neither is cure. However some factors such as apoptosis free radicals, heredity can partly explain the mechanism of AD. Some scholars have observed that the Ach inhibitor drugs and intelligence drugs can reduce the deadened of AD patients. As we all know AD can't be cured now, the patients must eat this western medicine for a long time. It is impotent for medical workers to find ways to cure this disease.
Some researches show that traditional Chinese medicine can slow the course of AD pathogenesis. Due to the seriousness side-effect of the western medicine, and the over all concept, the complexity, the diversity of the mechanism, traditional Chinese medicine has great advantages in the therapy of AD in the future.
DangguiShaoyao San (DSS) is a traditional Chinese medicine which was record in Synopsis of the Golden Chamber. DSS include six Medicinal herbs: ANGELICAESINENSISRADIX, PAEONIAERADIXALBA, CHUANXIONGRHIZOMA, PORIA, ATRACTYLODIS MACROCEPHALAERHIZOMA, ALISMATISRHIZOMA. ANGELICAESINENSISRADIX can add and activation the blood. PAEONIAERADIXALBA can relieve the pain and moderate the liver. DSS can nourish the blood and liver, maintain spleen and reduce moisture et, al. DSS which been record in Synopsis of the Golden Chamber often used to cure the pain on woman such as pregnant. Turn to cure AD patients effectively.
Previous research foundation of DSS:with the China National Science Foundation science research project "Danggui Shaoyao San chemical composition, dosage and effect relationship" support, we use the modern pharmacology research methods to research DSS of anti-dementia effects. The classic animal model induced by D-galactose induced subacute aging mice, and we observe the effects of DSS and single herb extract on model animal nervous system. The results showed that DSS San can remarkably improve the learning and memory ability of the model animal.
Traditional Chinese medicine is a major means of TCM clinical treatment of diseases, the reasonable combination of drugs can make to the overall effect, an optimal combination. DSS of senile dementia and its role in medicinal substances have close relations, and non-chemical these herbs in the sum of. Based on the above reasons, research and design will be combined with traditional Chinese medicine chemistry, quality control and pharmacological technology closely, to determine the material basis of DSS anti-senile dementia effect.
Objective:
Under the guidance of senile dementia pharmacological activity, we observe the relationship including DSS pharmacodynamic substances extraction, chemical composition, content analysis, fingerprinting, animal body chemical composition, aim to provide scientific basis of DSS.
Methods:
We screening pharmacological of anti-senile between DSS water extract to DSS alcohol extract by experimental method in pharmacology. Research establish subacute aging mice model which induced by D-galactose combined with AlCl3, to investigate the changes of body weight of mice, each group of mice was measured of working memory errors, reference memory errors, total errors in Eight Arms Maze.
Use the contents of Paeoniflorin, Ferulic acid, Chuanxiongzine hydrochloridum, Atractylenolide-1, Ligustilide as index, choosing solvent content, extracting times, extracting duration, solvent volume as influential factors, the optimal extraction condition was studied by L9(34) orthogonal test.
Divide the alcohol extraction of DSS into four parts, including petrolem ether part (DSS-P), ethyl acetate part (DSS-E), n-butyl alcohol part (DSS-N) and water part(DSS-W) by systematic solvent. Use HPLC-DAD method to determination the content of the index Component (Paeoniflorin, Ferulicacid, Chuanxiongzine hydrochloridum, Atractylenolide-1, Ligustilide) in DSS. Chromatographic conditions:SyncronisC18column (4.6mm×250mm,5μm) was used with the column temperature at30℃; the detection wavelength was set at220nm,270nm and320nm, and the injection volume was10μL at1.0mL/min flow rate and a gradient solution of acetonitrile and H2O (acidified to0.1%with phosphoric acid). Adopt HPLC-DAD method and multiwavelength Fusion Technology to establish the fingerprint of DSS, which are the related compounds, and using the Similarity Calculation Software of the chromatographic Fingerprint soft to evaluate the fingerprint.
We screening pharmacological of anti-senile (DSS, DSS-P, DSS-E, DSS-N, DSS-W) by experimental method in pharmacology.(1) Study the effects of DSS to the memory loss of AD-.mice caused by D-gal joint AlCl3, and further expound the material basis of DSS. Mice were randomly divided into control group, model group, Piracetam group, DSS group, DSS-P group, DSS-E group, DSS-N group, DSS-W group. Except control group, either group was injected with D-gal joint AlCl3to establish an aging model. The learning and study ability of mice was determined by Eight-arm radial maze test. The Body Mass index of the mice and the content of SOD, MDA, GSH-Px in serum, and the lipofuscin content in brain tissue were also determined by Eight-arm radial maze test. We observed the organization morphological changes in brain tissue with Eight-arm radial maze test.(2) Determined the antioxidant activity of the DSS group, DSS-P group, DSS-E group, DSS-N group, DSS-W group by radical DPPH systematic method, compared with ascorbic acid.(3) We select dopaminergic neurons have many similar characteristics of PC12cells as the experimental object, using hydrogen (H2O2) peroxide PC12cells damage induced by oxidative stress to establish the AD model of dopaminergic cells in vitro. By150,200,250,300,350,400,450μg/ml concentrations of DSS respectively, the cell survival rate was measured by MTT method. Study on the effect of compound hydrogen peroxide (H2O2) induced by PC12cell activity, different doses of compound (100,150,200,250,300,350,400μg/ml) preincubation of PC12cells with2h and H2O2, the survival rate of cells was determined by MTT method; the equivalent dose (300μg/ml) in various parts of the preincubation of PC,2cells with2h, and H2O26h coculture, cell survival rate was measured by MTT method.
We analyzed index compound in blood and tissues of rats by LC/MS/MS technique. Observe the changes of chemical components in the metabolic spectrum.(1) After given a one-time DSS (18g (herb drug)/kg) to SD rats, we take the blood samples at different time points. Research established LC/MS/MS method to measure the Paeoniflorin and Ferulicacid in Rat Plasma, which used to research for the drugs of the compound angelica peony bulk active ingredients in rats dynamics. Used the pharmacokinetics software in accordance with the non-compartment model to calculate the pharmacokinetic parameters and fitting the plasma concentration-time curve.(2)A single oral administration of compound (18g (herb drug)/kg), established the LC/MS/MS DSS multi-component rat tissue sample analysis method, and the quantitative analysis. Observe the changes of drug concentration distribution characteristics and organization that compound of multiple components in different tissues with time.
Statistical data were analyzed with SPSS13.0software processing, significant level from (α=0.05)(p＜0.05), had significant difference.
Results:
Induced aging mice by D-galactose combined with AlCl3, we observe that:after42days, compare with model group DSS alcohol group(high and low dose) and DSS water group (high and low dose) can add the mice's weight (p＜0.05). Results detection behavior:compare with model group DSS alcohol group(high and low dose) and DSS water group (high dose) can reduce working-time (P＜0.05).memory errors, reference memory errors, total errors (p＜0.05) in Eight Arms Maze. DSS alcohol group (high dose) show best effect.
Results of L9(34) orthogonal test:the optimal extraction condition of DSS was refluxing extraction for70%alcohol,2times and3h each time, alcohol volume8amount of herb drugs
Use HPLC-DAD method to develop the content determination method of the index Component in DSS. There were good linearity between the peak area and concentration when the amount is3.46～34.6μg for Paeoniflorin,0.174～ 1.74μg for Ferulicacid,5.1～51.0μg for Chuanxiongzine hydrochloridum,0.952～9.52μg for Atractylenolide-1,8.24～82.4μg for Ligustilide. The correlation coefficient of each component is0.9996,0.9999,0.9994,0.9990,0.9992respectively. Established three wavelength of the DSS (220nm,270nm,320nm) fusion fingerprint, calibration of5standard chromatographic peaks, the calibration of32characteristic peaks, degree of separation is good, good reproducibility and stability.
We screening pharmacological of anti-senile (DSS, DSS-P, DSS-E, DSS-N, DSS-W) by experimental method in pharmacology.(1) No7d, every group's weight is add (p＜0.05). No14d, Piracetam group's weight is increase (p＜0.01). No21d, compare with control group's weight, model group's weight is reduce (p＜0.01); compare with model group's weight, every administration group's weight is increase (p＜0.05). No28d, compare with control group' sweight, model group's weight is reduce(p＜0.01); compare with model group's weight, every administration group's weight is increase (p＜0.01or p＜0.05). No42d, compare with control group's weight, model group's weight is reduce (p＜0.01); compare with model group's weight, every administration group's weight is increase (p＜0.01or p＜0.05). Results detection behavior:compare with control group, model group add working-time, memory errors, reference memory errors, total errors (p＜0.01) in Eight Arms Maze. DSS and DSS-E, DSS-N group's result show best effect. Biochemical indexes (SOD, MDA, GSH-Px, lipofuscin) detection results show: compare with control group's SOD, GSH-Px, model group's SOD, GSH-Px is reduce (p＜0.01);compare with model group's SOD, GSH-Px, every administration group's SOD, GSH-Px is increase (p＜0.01or p＜0.05), but DSS and DSS-E group's result show best effect. Compare with control groups MDA and lipofuscin, model group's MDA and lipofuscin is increase (p＜0.01); compare with model group's MDA and lipofuscin, every administration group's MDA and lipofuscin is reduce (p＜0.01or p＜0.05), but DSS and DSS-E group's result show best effect. The general morphological observation under optical microscope HE staining:①control group's cortical cell structure is normal, cell nuclei clear, rich in cytoplasm, interstitial edema-free, glial cells are distributed between nerve cells in the hippocampus; pyramidal cell layer closely arranged, round and large nucleus, clear nucleolus②Model group's cortical cells and glial cells, reduce the cell are sparse, disordered, some nucleus pyknosis, cell line is not clear, nucleolus disappear, cytoplasm and hyperchromatic; hippocampal pyramidal cells reduced, are sparse and irregular, cell size, karyopyknosis phenomenon.③Compared with the model group, DSS, DSS-E, DSS-N group than in group improved significantly, cell structure of cortex of mice were arranged in order, close, nucleus visible; cortical areas still have varying degrees of nerve cell degeneration, but the number of nerve cell degeneration significantly reduced, less visible part of damaged cells, cell morphology tended to be normal, hippocampal pyramidal cell morphology, arrangement were normal.
(2) The DPPH scavenging rate curves show (ICS50): DSS, DSS-E part, DSS-N part, DSS-W part:0.3518,1.012,1.133,1.371mg/mL, Scavenging activity of radical DPPH by the acetate part of extracts from the DSS is the strongest, DSS-E part, DSS-N part, DSS-W part, DSS-P part successively followed.
(3) Use hydrogen peroxide (H2O2) to damage PC12cells,400,450μg/ml concentrations of DSS show cell toxicity. The survival rate of cells was120%、128%、129%、126%、100%、59%、51%(R＜0.05) by different doses of DSS (100,150,200,250,300,350,400μg/ml); The equivalent dose of compound (300μg/ml) parts could antagonize the action of H2O2, the cell survival rate was significantly increased compared with the model group, the difference was statistically significant (P<0.05), but no significant difference between various parts.
Analyze index DSS in blood and tissues of rats by LC/MS/MS technique.
(1) The experimental determination of paeoniflorin, ferulic acid content at different time in the plasma by LC/MS/MS method, which has high sensitivity, rapid, simple, accurate, short analysis time, peak is in5minutes, good reproducibility, and can be used for quantitative paeoniflorin, ferulic acid in rat plasma. But Ligustrazine,23-acetyl Alisol components was not detected, probably because the method sensitivity is not high enough, or it may be these components in rats in vivo into further study is needed to other substances.
Pharmacokinetic parameter show that paeoniflorin in vivo removed rapidly, the half-life is5±1.4h, curve has the double peak phenomenon (Cmax in the first0.5h), second peak occurred at about8-10h (concentration lower than first peak). The dosage and the area under the concentration-time curve of rats, the peak concentration was nonlinear correlation, suggesting paeoniflorin in dynamical behavior of nonlinear dynamics process of rats in vlvo.
(2) Rats were gavaged with DSS, in rat's vivo tissues distribution of high order as follows:
15min in the rat sequence of paeoniflorin concentration order:stomach> liver> lung>intestine> heart> blood> kidney> brain;
35min in the rat sequence of paeoniflorin concentration order:stomach) blood> lung>intestine> kidney> liver> heart> brain;6h in the rat sequence of paeoniflorin concentration order:stomach) blood>intestine> kidney> liver> lung)heart> brain.
15min in rat tissues of ferulic acid concentration order:stomach>intestine> liver> lung> heart> blood)kidney> brain;
35min in rat tissues of ferulic acid concentration order:stomach>intestine) lung>brain> liver) kidney> blood> heart;
6h in rat tissues of ferulic acid concentration order:stomach>intestine> lung> kidney> blood.
Conc Iusions:
Study on the comprehensive use of chemical fingerprint, pharmacodynamics, pharmacokinetics, and joint research pattern that Danggui Shaoyao San mechanism of anti-aging.
Chemical fingerprint:using the HPLC-DAD method of Danggui Shaoyao San and petroleum ether extract, ethyl acetate, n-butanol and water area, part of the main components of paeoniflorin, ferulic acid, ligustrazine, atractylenolide I, the content of ligustilide. Found that the content of each component of Danggui Shaoyao San and in different parts of each level. Established the Danggui Shaoyao San three wavelength (220nm,270nm,320nm) fusion fingerprint, calibration of5control of paeoniflorin, ferulic acid, ligustrazine, atractylenolide I, ligustilide chromatographic peak, the calibration of32characteristic peaks, degree of separation is good, good reproducibility and stability, provide quality guarantee for the pharmacodynamic study.
Pharmacodynamics experiment demonstrated that abnormal and spatial cognitive Danggui Shaoyao San can obviously improve the behavior induced by D-galactose combined with A1Cl3in subacute aging mice, improve the ability of learning and memory; reduce oxidative free radical damage to brain tissue; abnormal tissue caused by improving the pathological factors in experimental animal brain. In vitro antioxidant proved its antioxidant capacity size order: Danggui Shaoyao San, ethyl acetate, n-butanol and water position, petroleum ether fraction. PC12cells in vitro experiments prove that Danggui Shaoyao San and each position can antagonize the action of H2O2, the cell survival rate was significantly increased, but no significant difference between various parts, likely Danggui Shaoyao San showed a protective effect on nerve cells in the overall point of view.
Study on pharmacokinetics showed that rat's plasma paeoniflorin ferulic acid is the main effective components of Danggui Shaoyao San antidementia activity points. Given to rats orally Danggui Shaoyao San, Danggui Shaoyao San can be rapidly distributed in the body most organs, organizations of paeoniflorin and ferulic acid concentration reached the peak of rat5min, Danggui Shaoyao San in rats of paeoniflorin and ferulic acid were the highest content of gastrointestinal tract, the brain with minimum quantity, relative paeoniflorin, ferulic acid elimination rate slow.
Chemical composition and pharmacodynamic study of Danggui Shaoyao San shows that the analysis of Pearson and grey correlation degree:in front of the5known index component of paeoniflorin, ferulic acid, ligustrazine, ligustilide, atractylenolide I and efficacy of highly correlated, scientific combination of pharmacokinetic results in the experiment the mechanical angle5the index components as the pharmacodynamic material bases.
随着人类寿命的延长,全球进入老龄化社会。目前AD已成为继癌症、心血管疾病和中风之后威胁老年人健康的又一大疾病。我国的流行病学调查资料显示,AD在65岁以上人群中的发病率为3%-5%,接近西方发达国家水平。衰老、基因突变及遗传为AD明确的致病原因,此外炎症及应激,神经递质减少,代谢异常等都为其可能的致病原因[2-3]。
AD至今无有效的治疗方法,目前主要是对症治疗。多年以来,乙酰胆碱酯酶抑制剂如加兰他敏、石杉碱甲,谷氨酸受体调控剂如盐酸美金刚胺,脑循环改善剂如尼麦角林等,γ-氨基丁酸类促智药如吡拉西坦等被广泛应用于AD的治疗[4-7]。但由于AD病人需要长期服药,西药的副作用比较大,不断寻求副作用小的药物以有效地治疗AD成为现代社会人们广泛关注的重要问题。脏腑失调,气血亏虚,脑髓失养是形成AD的必要条件,肾之精气化精生髓汇聚而为脑,五脏气血之精华皆上荣于脑,脑髓之功能发挥,须依赖气血滋养。老年人脏腑功能失调,气血生化乏源,脑髓得不到充足的气血滋养,久则神明受损,功能低下。中药当归芍药散具有整体作用的特点,与临床常用的抗老年性痴呆化学药物相比,它们的副作用比较小,不易产生耐药性,有明显前景和优势[8-10]。当归芍药散(DangguiShaoyao San,以下简称DSS)是《金匮要略》所载著名方剂[1],由当归、芍药、川芎、茯苓、白术、泽泻(1：5.3：2.6：1.3：1.3：2.6)六味药组成。当归具有补血活血,调经止痛,润肠通便的功效。芍药有养血敛阴,补而不腻,柔肝缓中,止痛收汗功效。当归芍药散具有养血调肝,健脾利湿,养血益脾等功效。原方主治“妇人妊娠诸疾痛”以及“妇人杂病腹中诸疾痛”。目前有研究将DSS转变为治疗老年性痴呆的有效方剂,疗效确切[12-14]。前期的研究基础：本课题在国家自然基金“当归芍药散化学成分、剂量配比与药效学关系”的研究项目资助的支持下,采用现代药理研究方法,研究当归芍药散的抗痴呆作用。采用D-半乳糖致致小鼠亚急性衰老的经典动物模型,考察了当归芍药散及单味药材提取液对动物神经系统的影响[15-17],结果发现当归芍药散能显著改善学习记忆能力。中药方剂是中医临床治疗疾病的主要手段,药物的合理配伍可以相使相须,达到一种优化组合的整体疗效。当归芍药散的抗老年痴呆作用与其组方药材有着密切的关系,又非这些药材中化学成分的简单加和。基于以上原因,研究设计将中药化学、质量控制以及药理学技术紧密结合,以确定当归芍药散抗老年痴呆作用的物质基础。
目的
在抗老年痴呆药理活性指导下,对当归芍药散(DSS)(当归、芍药、川芎、茯苓、白术、泽泻(1：5.3：2.6：1.3：1.3：2.6))药效物质的提取工艺、化学成分及其含量分析、指纹图谱、化学成分在动物体内代谢等进行了相关研究,旨在为当归芍药散的二次开发提供科学依据。
方法
采用药理学实验研究方法,对当归芍药散水提和醇提部位进行了抗老年痴呆的药理筛选研究。建立D-半乳糖联合AlCl3所致亚急性衰老小鼠的模型,考察各组小鼠体重变化,统计各组小鼠八臂迷宫的工作记忆错误次数、参考记忆错误次数、错误总数次数。
采用Lg(34)正交表对抗痴呆药效最好的当归芍药散醇提部位(以下简称当归芍药散)的提取工艺进行了研究。因素选择：乙醇浓度(A)、提取次数(B)、提取时间(C)、乙醇用量(D),每个因素设置3个水平。选择多指标成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行综合测评,确定各因素的最佳提取工艺水平。在此基础上,采用系统溶剂分离法,对当归芍药散(DSS)进行了部位分离,分离了石油醚部位(DSS-P)、乙酸乙酯部位(DSS-E)、正丁醇部位(DSS-N)、水部位(DSS-W),以进行化学成分和药效学研究。
建立了当归芍药散(DSS)及4个部位的主要活性成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行了HPLC-DAD含量分析方法。色谱条件为：色谱柱syncronisC18(4.6mm×250mm,5μm)不锈钢柱；流动相：A：乙腈,B：0.1%磷酸,梯度程序条件洗脱；流速：1.Oml/min;柱温：30C°；进样体积：10μL；用二极管检测器进行190nm-400nm全波长扫描,经比较后发现220nm、270nm、320nm处基线平稳,出峰多,峰强度较好,故确定检测波长为220nm、270nm、320nm。λ1=220nm(检测芍药苷),λ2=270nm(检测川芎嗪、白术内酯Ⅰ),λ3=320nm(检测藁本内酯、阿魏酸。对当归芍药散进行了指纹图谱的研究,标定了共有峰,采用中药色谱指纹图谱相似度评价系统进行相似度分析,建立当归芍药散的三波长融合指纹图谱,使用化学对照品和药材对色谱峰进行定性、归属。
药效试验采用体内结合体外研究方法,对当归芍药散(DSS)及石油醚部位(DSS-P)、乙酸乙酯部位(DSS-E)、正丁醇部位(DSS-N)、水部位(DSS-W)进行了抗痴呆筛选研究。(1)利用D-半乳糖联合AlCl3造成小鼠老年痴呆病理的复合模型,出现学习记忆力减退；海马和皮质老年斑形成,促进Aβ的沉积等类似AD病变,造模机制比较接近AD。用八臂迷宫研究动物的行为学变化,检测其生化指标(SOD、MDA、GSH-Px、脂褐素),HE染色观察脑组织病理形态学等方面对当归芍药散及四个部位进行了研究；(2)采用体外清除1,1-二苯基苦基苯肼(DPPH)法对当归芍药散(DSS)及其部位进行抗氧化研究。当有自由基清除齐(?)DPPH存在时,由于与DPPH的单电子配对而使其吸收逐渐消失,其褪色程度与配对电子数成化学计量关系,因而可用分光光度法进行定量分析；(3)选用与多巴胺能神经元有许多相似特征的PC12细胞为实验对象,以过氧化氢(H2O2)诱导PC12细胞损伤,从而建立体外多巴胺能细胞的AD氧化应激模型,研究当归芍药散(DSS)及部位的保护作用,包括：H2O2作用时间和浓度的选择、当归芍药散(DSS)对细胞毒性考察和浓度的选择、当归芍药散(DSS)对细胞存活率的影响。用倒置显微镜观察培养的PC12细胞的形态,用形态学方法证明PC12细胞培养成功。采用150、200、250、300、350、400、450μg/ml浓度的当归芍药散(DSS)分别与PC12细胞共同孵育24h,以MTT法测定细胞存活率。考察当归芍药散对过氧化氢(H202)诱导PC12细胞活性的影响,不同剂量当归芍药散(100、150、200、250、300、350、400μg/ml预孵育PC12细胞2h,再和H2O2共培养6h,以MTT法测定细胞存活率；采用剂量相当于当归芍药散(DSS)(300μg/ml)的各个部位预孵育PC,2细胞2h,再和H2O2共培养6h,以MTT法测定细胞存活率。(4)当归芍药散(DGS)的化学成分与药效相关性研究,利用本章药效实验数据与第二章质量控制的HPLC实验数据,将当归芍药散、乙酸乙酯、正丁醇、水部位得到的化学成分对应的色谱图中各色谱峰Xi与本章药效实验数据(变量Y(Y1：小鼠42天体重增长鼠；Y2：八臂迷宫RME错误次数；Y3：八臂迷宫WME错误次数； Y4：八臂迷宫错误总次数；Y5：八臂迷宫时间；Y6：小鼠血浆SOD含量；Y7：小鼠血浆MDA含量；Y8：小鼠血浆GSH-Px含量；Y9：小鼠脑内脂褐素的含量；Y10：对DPPH清除的影响；Y11：对PC12的存活率))进行相关性统计分析,研究当归芍药散化学成分与药效之间的关系。
基于LC/MS/MS技术分析当归芍药散在大鼠血液和组织中指标性成分,观察其代谢的化学成分谱变化。(1)建立的多成分LC/MS/MS法分析方法学认证的基础上,对单次口服当归芍药散(18g(生药)/Kg)后的大鼠血浆中多种成分的药代动力学进行了初步的研究,考察成分的药代动力学参数。色谱及质谱条件色谱柱为Eclipse XDB C18柱(3.5μm,2.0mm×150mm),流动相为甲醇：10mmol·L-1乙酸铵(90：10),流速为200μL·min-1,样品进样量为20μL,柱温：35℃,分析时间为3.0min,用外标法进行定量分析。采用辅助气化电喷雾离子源(ESI)正离子和负离子分时段监测模式(MRM)进行定量分析,0-2.8min时段负离子监测离子对为：m/z478.9→448.9(芍药苷)和m/z192.8→177.8(阿魏酸),2.8-6min时段正离子监测离子对为：m/z231.4→185.1(白术内酯-Ⅰ)。离子源的雾化气和气帘气均为氮气,流量分别为70、30mL·min-1、碰撞气(氮气)流量为12L·min-1,离子源喷雾电压：-4500V/+4500V,雾化温度：450℃。(2)单次口服当归芍药散(18g(生药)/Kg)后,建立了大鼠的组织样品中当归芍药散多成分的LC/MS/MS分析方法,灌胃当归芍药散给大鼠后,参考芍药苷和阿魏酸血药浓度—时间曲线,确定采样时间点为15min,35min和6h。SD大鼠15只,每组5只,随机分为3组。灌胃当归芍药散后分别于给药后的15min,35min和6h,将大鼠迅速颈椎脱臼处死,分离胃,肠,肺,肾,脑,心,肝,脾等组织(胃,肠组织分别用蒸馏水将内容物冲洗于净,用干燥滤纸吸干),共8种组织。分别进行LC/MS/MS定量分析。阐明当归芍药散多成分在各组织中的分布特点及组织中药物浓度随时间的变化规律。
统计数据均用SPSS13.0统计分析软件处理,显著性水平取α=0.05(双侧),P<0.05为差异有统计学意义。
结果
1.提取工艺的研究
在当归芍药散对D-半乳糖联合AlCl3所致亚急性衰老小鼠的影响实验中发现：42d检测行为学实验结果：与模型组比及当归芍药散醇提部位组(高、低剂量)和水提部位组(高、低剂量)均使小鼠在走完八臂迷宫所用时间、小鼠工作记忆错误、参考记忆错误、错误总数减少,有显著性差异(P<0.05)。各给药组间比较,当归芍药散醇提部位组高剂量(40g/Kg)(以下简称当归芍药散)表现最好。
在确认改善AD小鼠学习能力效果最好的是DSS乙醇浸膏高剂量的药效活性指导下,采用(L34)正交设计实验对当归芍药散的乙醇提取工艺进行了研究,选择多指标芍药苷、阿魏酸、白术内酯、川芎嗪、藁本内酯为定量指标进行综合测评。依据方差分析结果：A、B、D因素均对实验结果差异有显著性(P<0.05),A2优于A1和A3；B2优于B1和B3；D2优于D1和D3,各因素影响程度依次为A>B>D>C,再结合直观分析结果和实际情况综合考虑,确定各因素的最佳水平组合为A2B2C3D2,采用70%乙醇,提取2次,提取3小时,乙醇用量8倍。
2.当归芍药散质量控制的研究
采用HPLC-DAD法分别对当归芍药散(DSS)及石油醚部位(DSS-P)、乙酸乙酯部位(DSS-E)、正丁醇部位(DSS-N)、水部位(DSS-W)的主要活性成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行了含量分析研究,芍药苷lgy=0.97381gx+5.968,r=0.9996,含量在3.46-34.6μg范围内线性关系良好；阿魏酸1gy=1.01971gx+6.728,r=0.9999,含量在0.174-1.74μg1范围内线性关系良好；川芎嗪1gy=0.99731gx+6.105,r=0.9994,含量在5.1-51.0u g范围内线性关系良好；白术内酯Ⅰ1gy=0.16261gx+6.687,r=0.9990,含量在0.952-9.52μg范围内线性关系良好；藁本内酯1gy=0.98161gx+5.161,r=0.9992,含量在8.24-82.4μg范围内线性关系良好。其中当归芍药散中5种成分测定结果分别为：芍药苷5.945mg/g、阿魏酸0.042mg/g、川芎嗪3.930mg/g、白术内酯Ⅰ0.7200mg/g、藁本内酯1.470mg/g；石油醚部位中白术内酯10.1790mg/g、藁本内酯0.8770mg/g；乙酸乙酯部位中芍药苷1.261mg/g、阿魏酸0.037mg/g、川芎嗪3.045mg/g；正丁醇部位中芍药苷2.801mg/g、阿魏酸0.0040mg/g、川芎嗪0.9940mg/g；水部位中芍药苷0.2080mg/g、川芎嗪2.497mg/g。5种成分的含量特点：芍药苷：DSS>DSS-N>DSS-E>DSS-W:阿魏酸：DSS>DSS-E>DSS-N:川芎嗪：DSS>DSS-E>DSS-W>DSS-N>DSS-P:白术内酯Ⅰ：DSS>DSS-P:藁本内酯：DSS>DSS-P。
建立了该当归芍药散的三波长(220nm,270nm,320nm)融合指纹图谱,10批样品230nm指纹图谱有30个共有峰,270nm处有25个共有峰,320nm处有20个共有峰。三波长融合指纹图谱共有32个共有峰,在峰数及含量上兼顾三个特征波长指纹的特点。标定5个对照品芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯的色谱峰,标定了共有峰32个,方法分离度好,重现性和稳定性良好。其中0-68min为220nm波长下的指纹图谱、270nm波长下的指纹图谱,68-90min为320nm波长下的指纹图谱。3.药效试验采用体内结合体外研究方法,对当归芍药散及石油醚部位、乙酸乙酯部位、正丁醇部位、水部位进行了抗衰老筛选研究。
(1)第7d各组小鼠体质量增长数有明显统计学差异(P<0.05)。第14d与阳性组比模型组的质量增长数显著性增加(P<0.01),第21d时,与空白组比较,模型组体质量增长数减少(P<0.01)；与模型组比较,当归芍药散及各部位组体质量增长数增加(P<0.05);42d时与空白组比,模型组体质量增长数减少,有明显统计学差异(P<0.01),与模型组比较,当归芍药散组及各部位组体质量增长数增加(P<0.01或P<0.05)。行为学实验结果：模型组小鼠走完八臂迷宫所用时间、小鼠工作记忆错误次数、参考记忆错误次数、错误总次数高于空白组(P<0.01),造模成功；当归芍药散组及石油醚部位、乙酸乙酯部位、正丁醇部位组小鼠走完八臂迷宫所用时间、工作记忆错误、参考记忆错误、错误总数少于模型组(P<0.01),各给药组间比较,各部位组中乙酸乙酯组和正丁醇组为最好。生化指标(SOD、MDA、GSH-Px、脂褐素)检测结果表明：与空白组比模型组SOD活性明显降低(P<0.01)。与模型组比各个部位明显升高损伤的小鼠血中SOD活性,尤其以当归芍药散和乙酸乙酯部位最好。与空白组比模型组MDA活性明显升高(P<0.01)。与模型组比各个部位明显升高损伤的小鼠血中MDA,差异均具有显著性意义(P<0.01)。尤其以当归芍药散和乙酸乙酯部位最好。与空白组比模型组GSH-Px活性明显降低(P<0.01)。与模型组比各个部位明显升高损伤的小鼠血中GSH-Px活性,差异均具有非常显著性意义(P<0.01)。尤其以当归芍药散、乙酸乙酯部位、正丁醇部位最好。脑内脂褐素结果表明：与空白组比模型组脂褐素明显升高(P<0.01)；与模型组比当归芍药散及各部位明显降低小鼠脑中脂褐素的含量。HE染色的一般形态学光镜下观察：①空白组小鼠皮质区神经细胞结构正常,数量较多,胞核清晰,胞浆丰富,间质无水肿表现,胶质细胞散布于神经细胞之间；海马区锥体细胞层排列紧密,细胞核圆而大,核仁清晰。②模型组皮质区神经细胞减少和胶质细胞增多、细胞排列稀疏、杂乱、有的细胞核固缩、细胞线不清晰,核仁消失,胞浆深染；海马区锥体细胞层数减少,排列稀疏不规则,细胞体积变小,可见核固缩现象。③与模型组比当归芍药散组、乙酸乙酯组、正丁醇组比有明显改善,小鼠皮质区细胞结构排列较整齐、紧密、细胞核可见；皮质区仍有不同程度的神经细胞变性.但变性神经细胞数量明显减少,可见少部分损伤细胞,细胞形态趋于正常,海马区锥体细胞形态、排列基本正常。
(2)由当归芍药散及各部位对DPPH自由基的清除率作曲线可知,当归芍药散、乙酸乙酯、正丁醇、水部位提取物的IC50值分别为0.3518,1.012、1.133、1.371mg/mL,石油醚无IC50值。清除DPPH自由基能力的大小顺序依次为：当归芍药散>乙酸之酯部位>正丁醇部位>水部位>石油醚部位。但是溶液质量浓度在2mg/mL以上时当归芍药散与乙酸乙酯部位清除率接近。
(3)采用不同浓度的当归芍药散的结果发现：与正常细胞比当归芍药散在400、450μg/ml时对PC12细胞明显毒性作用(P<0.05)。不同浓度当归芍药散可拮抗H2O2的作用,细胞存活率明显升高,各剂量下细胞存活率分别为120%、128%、129%、126%、100%、59%、51%,具有明显的剂量相关性(P<0.001),与正常对照组比较差异均具有统计学意义(P<0.05)。采用剂量相当于当归芍药散(300μg/m1)的各部位均可拮抗H,O,的作用,细胞存活率明显升高,与模型组比较差异均具有统计学意义(P<0.05),但是各部位之间无明显差异,有可能当归芍药散在全方整体角度表现出对神经细胞的保护作用。(4)当归芍药散的化学成分与药效相关性研究发现Pearson分析和灰色关联度分析表明：前面所述5个已知指标性成分芍药苷,阿魏酸,川芎嗪,藁本内酯,白术内酯Ⅰ与药效具有高度相关性。
4.当归芍药散大鼠体内多成分的药代动力学研究本实验采用LC/MS/MS法测定各时间点血浆中芍药苷、阿魏酸的含量,灵敏度高、快速、简单、准确、分析时间短,在5分钟内出峰完毕,重现性好,可用于大鼠血浆中芍药苷、阿魏酸的定量,其它成分未能检出,可能是因为该测定方法灵敏度不够高,或可能是这些成分在大鼠体内转化成其他物质有待今后进一步研究。大鼠单次灌胃当归芍药散后,芍药苷的吸收和消除十分迅速,在给药后的血浆中能很快测出较高浓度的芍药苷(大鼠的Cmax出现在前0.5h),药-时曲线呈现双峰现象,约在8-10h出现第2个峰值(浓度低于第1个峰值)。表明该成分体内吸收较快,药物在体内消除较快,半衰期为5.0±1.4h。
大鼠灌胃当归芍药散后,在大鼠体内芍药苷和阿魏酸各组织分布高低顺序如下
(1)大鼠体内15min时各组织中芍药苷浓度顺序为：胃>肝>肺>肠>心>血>肾>>脑：
(2)大鼠体内35min时各组织中芍药苷浓度顺序为：胃>血>肺>肠>肾>肝>心>>脑：
(3)大鼠体内6h时各组织中芍药苷浓度顺序为：胃>血>肠>肾>肝≈肺≈心>脑。
(4)大鼠体内15min时各组织中阿魏酸浓度顺序为：胃>肠>肝>肺>心>血>肾≈脑：
(5)大鼠体内35min时各组织中阿魏酸浓度顺序为：胃>肠>肺>>脑>肝>肾>血>心：
(6)大鼠体内6h时各组织中阿魏酸浓度顺序为：胃>肠>肺>肾>血。
大鼠灌胃当归芍药散后,当归芍药散可迅速分布于全身大多数组织脏器,大鼠5min时各组织芍药苷和阿魏酸浓度达到峰值,这与血药浓度-时间曲线变化一致。6h时大鼠各组织中芍药苷和阿魏酸浓度降至不足峰值时的10%。当归芍药散在大鼠中芍药苷和阿魏酸均以胃肠道中含量最高,脑中含量最低,相对芍药苷来说阿魏酸的组织消除速率比较慢。
结论
研究综合采用化学指纹图谱、药效学、药代动力学等联合研究的模式证明了当归芍药散抗衰老的作用机制。
化学指纹图谱：采用HPLC-DAD法分别对当归芍药散及石油醚部位、乙酸乙酯部位、正丁醇部位、水部位的主要指标性成分芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯进行了含量分析研究。发现当归芍药散及各部位中各种成分的含量各有高低。建立了该当归芍药散的三波长(220nm,270nm,320nm)融合指纹图谱,标定5个对照品芍药苷、阿魏酸、川芎嗪、白术内酯Ⅰ、藁本内酯的色谱峰,标定了共有峰32个,方法分离度好,重现性和稳定性良好,为药效研究提供质量保证。
药效学实验：证明当归芍药散能明显改善D-半乳糖联合Alcl3所致亚急性衰老小鼠行为学的异常和空间认知障碍,提高学习记忆能力；减少自由基对脑组织的氧化损害;改善病理因素造成的实验动物脑内组织形态的异常。体外抗氧化实验证实其抗氧化能力的大小顺序依次为：当归芍药散>乙酸乙酯部位>正丁醇部位>水部位>石油醚部位。体外PC12细胞实验证明当归芍药散和各部位可拮抗H2O2的作用,细胞存活率明显升高,但是各部位之间无明显差异,有可能当归芍药散在整体角度表现出对神经细胞的保护作用。
药代动力学研究表明：大鼠体内血浆芍药苷阿魏酸等是当归芍药散抗痴呆活性的主要有效组分。给大鼠灌胃当归芍药散后,当归芍药散可迅速分布于全身大多数组织脏器,大鼠5min时各组织芍药苷和阿魏酸浓度达到峰值,当归芍药散在大鼠中芍药苷和阿魏酸均以胃肠道中含量最高,脑中含量最低,相对芍药苷来说阿魏酸的组织消除速率比较慢。
当归芍药散的化学成分与药效相关性研究发现Pearson分析和灰色关联度分析表明：前面所述5个已知指标性成分芍药苷,阿魏酸,川芎嗪,藁本内酯,白术内酯Ⅰ与药效具有高度相关性,结合药代动力学结果在实验角度阐明这5个指标性成分作为药效物质基础的科学性。
Alzheimer's disease (AD) is a kind of neuron degenerative disease that causes behavior deterioration and progressive cognitive. Although the causes of AD diverse are complex, but the histopathoiogical features are almost exactly the same, such as the formation of nerve cells outside the A β amyloidal protein, which was seen as a direct result in the pathological causes of AD, the formation of neuron fibril tangles in nerve cells, the feature of senile plaques, decrease in the number of nerve cells.
As the life of human being increase, AD often can find in the elderly over65more and more. The number of people with AD has increased too, which is heavy burden to AD patient's families and our society.
Currently there is no certain doctrine about etiology and pathogenesis of AD, neither is cure. However some factors such as apoptosis free radicals, heredity can partly explain the mechanism of AD. Some scholars have observed that the Ach inhibitor drugs and intelligence drugs can reduce the deadened of AD patients. As we all know AD can't be cured now, the patients must eat this western medicine for a long time. It is impotent for medical workers to find ways to cure this disease.
Some researches show that traditional Chinese medicine can slow the course of AD pathogenesis. Due to the seriousness side-effect of the western medicine, and the over all concept, the complexity, the diversity of the mechanism, traditional Chinese medicine has great advantages in the therapy of AD in the future.
DangguiShaoyao San (DSS) is a traditional Chinese medicine which was record in Synopsis of the Golden Chamber. DSS include six Medicinal herbs: ANGELICAESINENSISRADIX, PAEONIAERADIXALBA, CHUANXIONGRHIZOMA, PORIA, ATRACTYLODIS MACROCEPHALAERHIZOMA, ALISMATISRHIZOMA. ANGELICAESINENSISRADIX can add and activation the blood. PAEONIAERADIXALBA can relieve the pain and moderate the liver. DSS can nourish the blood and liver, maintain spleen and reduce moisture et, al. DSS which been record in Synopsis of the Golden Chamber often used to cure the pain on woman such as pregnant. Turn to cure AD patients effectively.
Previous research foundation of DSS:with the China National Science Foundation science research project "Danggui Shaoyao San chemical composition, dosage and effect relationship" support, we use the modern pharmacology research methods to research DSS of anti-dementia effects. The classic animal model induced by D-galactose induced subacute aging mice, and we observe the effects of DSS and single herb extract on model animal nervous system. The results showed that DSS San can remarkably improve the learning and memory ability of the model animal.
Traditional Chinese medicine is a major means of TCM clinical treatment of diseases, the reasonable combination of drugs can make to the overall effect, an optimal combination. DSS of senile dementia and its role in medicinal substances have close relations, and non-chemical these herbs in the sum of. Based on the above reasons, research and design will be combined with traditional Chinese medicine chemistry, quality control and pharmacological technology closely, to determine the material basis of DSS anti-senile dementia effect.
Objective:
Under the guidance of senile dementia pharmacological activity, we observe the relationship including DSS pharmacodynamic substances extraction, chemical composition, content analysis, fingerprinting, animal body chemical composition, aim to provide scientific basis of DSS.
Methods:
We screening pharmacological of anti-senile between DSS water extract to DSS alcohol extract by experimental method in pharmacology. Research establish subacute aging mice model which induced by D-galactose combined with AlCl3, to investigate the changes of body weight of mice, each group of mice was measured of working memory errors, reference memory errors, total errors in Eight Arms Maze.
Use the contents of Paeoniflorin, Ferulic acid, Chuanxiongzine hydrochloridum, Atractylenolide-1, Ligustilide as index, choosing solvent content, extracting times, extracting duration, solvent volume as influential factors, the optimal extraction condition was studied by L9(34) orthogonal test.
Divide the alcohol extraction of DSS into four parts, including petrolem ether part (DSS-P), ethyl acetate part (DSS-E), n-butyl alcohol part (DSS-N) and water part(DSS-W) by systematic solvent. Use HPLC-DAD method to determination the content of the index Component (Paeoniflorin, Ferulicacid, Chuanxiongzine hydrochloridum, Atractylenolide-1, Ligustilide) in DSS. Chromatographic conditions:SyncronisC18column (4.6mm×250mm,5μm) was used with the column temperature at30℃; the detection wavelength was set at220nm,270nm and320nm, and the injection volume was10μL at1.0mL/min flow rate and a gradient solution of acetonitrile and H2O (acidified to0.1%with phosphoric acid). Adopt HPLC-DAD method and multiwavelength Fusion Technology to establish the fingerprint of DSS, which are the related compounds, and using the Similarity Calculation Software of the chromatographic Fingerprint soft to evaluate the fingerprint.
We screening pharmacological of anti-senile (DSS, DSS-P, DSS-E, DSS-N, DSS-W) by experimental method in pharmacology.(1) Study the effects of DSS to the memory loss of AD-.mice caused by D-gal joint AlCl3, and further expound the material basis of DSS. Mice were randomly divided into control group, model group, Piracetam group, DSS group, DSS-P group, DSS-E group, DSS-N group, DSS-W group. Except control group, either group was injected with D-gal joint AlCl3to establish an aging model. The learning and study ability of mice was determined by Eight-arm radial maze test. The Body Mass index of the mice and the content of SOD, MDA, GSH-Px in serum, and the lipofuscin content in brain tissue were also determined by Eight-arm radial maze test. We observed the organization morphological changes in brain tissue with Eight-arm radial maze test.(2) Determined the antioxidant activity of the DSS group, DSS-P group, DSS-E group, DSS-N group, DSS-W group by radical DPPH systematic method, compared with ascorbic acid.(3) We select dopaminergic neurons have many similar characteristics of PC12cells as the experimental object, using hydrogen (H2O2) peroxide PC12cells damage induced by oxidative stress to establish the AD model of dopaminergic cells in vitro. By150,200,250,300,350,400,450μg/ml concentrations of DSS respectively, the cell survival rate was measured by MTT method. Study on the effect of compound hydrogen peroxide (H2O2) induced by PC12cell activity, different doses of compound (100,150,200,250,300,350,400μg/ml) preincubation of PC12cells with2h and H2O2, the survival rate of cells was determined by MTT method; the equivalent dose (300μg/ml) in various parts of the preincubation of PC,2cells with2h, and H2O26h coculture, cell survival rate was measured by MTT method.
We analyzed index compound in blood and tissues of rats by LC/MS/MS technique. Observe the changes of chemical components in the metabolic spectrum.(1) After given a one-time DSS (18g (herb drug)/kg) to SD rats, we take the blood samples at different time points. Research established LC/MS/MS method to measure the Paeoniflorin and Ferulicacid in Rat Plasma, which used to research for the drugs of the compound angelica peony bulk active ingredients in rats dynamics. Used the pharmacokinetics software in accordance with the non-compartment model to calculate the pharmacokinetic parameters and fitting the plasma concentration-time curve.(2)A single oral administration of compound (18g (herb drug)/kg), established the LC/MS/MS DSS multi-component rat tissue sample analysis method, and the quantitative analysis. Observe the changes of drug concentration distribution characteristics and organization that compound of multiple components in different tissues with time.
Statistical data were analyzed with SPSS13.0software processing, significant level from (α=0.05)(p＜0.05), had significant difference.
Results:
Induced aging mice by D-galactose combined with AlCl3, we observe that:after42days, compare with model group DSS alcohol group(high and low dose) and DSS water group (high and low dose) can add the mice's weight (p＜0.05). Results detection behavior:compare with model group DSS alcohol group(high and low dose) and DSS water group (high dose) can reduce working-time (P＜0.05).memory errors, reference memory errors, total errors (p＜0.05) in Eight Arms Maze. DSS alcohol group (high dose) show best effect.
Results of L9(34) orthogonal test:the optimal extraction condition of DSS was refluxing extraction for70%alcohol,2times and3h each time, alcohol volume8amount of herb drugs
Use HPLC-DAD method to develop the content determination method of the index Component in DSS. There were good linearity between the peak area and concentration when the amount is3.46～34.6μg for Paeoniflorin,0.174～ 1.74μg for Ferulicacid,5.1～51.0μg for Chuanxiongzine hydrochloridum,0.952～9.52μg for Atractylenolide-1,8.24～82.4μg for Ligustilide. The correlation coefficient of each component is0.9996,0.9999,0.9994,0.9990,0.9992respectively. Established three wavelength of the DSS (220nm,270nm,320nm) fusion fingerprint, calibration of5standard chromatographic peaks, the calibration of32characteristic peaks, degree of separation is good, good reproducibility and stability.
We screening pharmacological of anti-senile (DSS, DSS-P, DSS-E, DSS-N, DSS-W) by experimental method in pharmacology.(1) No7d, every group's weight is add (p＜0.05). No14d, Piracetam group's weight is increase (p＜0.01). No21d, compare with control group's weight, model group's weight is reduce (p＜0.01); compare with model group's weight, every administration group's weight is increase (p＜0.05). No28d, compare with control group' sweight, model group's weight is reduce(p＜0.01); compare with model group's weight, every administration group's weight is increase (p＜0.01or p＜0.05). No42d, compare with control group's weight, model group's weight is reduce (p＜0.01); compare with model group's weight, every administration group's weight is increase (p＜0.01or p＜0.05). Results detection behavior:compare with control group, model group add working-time, memory errors, reference memory errors, total errors (p＜0.01) in Eight Arms Maze. DSS and DSS-E, DSS-N group's result show best effect. Biochemical indexes (SOD, MDA, GSH-Px, lipofuscin) detection results show: compare with control group's SOD, GSH-Px, model group's SOD, GSH-Px is reduce (p＜0.01);compare with model group's SOD, GSH-Px, every administration group's SOD, GSH-Px is increase (p＜0.01or p＜0.05), but DSS and DSS-E group's result show best effect. Compare with control groups MDA and lipofuscin, model group's MDA and lipofuscin is increase (p＜0.01); compare with model group's MDA and lipofuscin, every administration group's MDA and lipofuscin is reduce (p＜0.01or p＜0.05), but DSS and DSS-E group's result show best effect. The general morphological observation under optical microscope HE staining:①control group's cortical cell structure is normal, cell nuclei clear, rich in cytoplasm, interstitial edema-free, glial cells are distributed between nerve cells in the hippocampus; pyramidal cell layer closely arranged, round and large nucleus, clear nucleolus②Model group's cortical cells and glial cells, reduce the cell are sparse, disordered, some nucleus pyknosis, cell line is not clear, nucleolus disappear, cytoplasm and hyperchromatic; hippocampal pyramidal cells reduced, are sparse and irregular, cell size, karyopyknosis phenomenon.③Compared with the model group, DSS, DSS-E, DSS-N group than in group improved significantly, cell structure of cortex of mice were arranged in order, close, nucleus visible; cortical areas still have varying degrees of nerve cell degeneration, but the number of nerve cell degeneration significantly reduced, less visible part of damaged cells, cell morphology tended to be normal, hippocampal pyramidal cell morphology, arrangement were normal.
(2) The DPPH scavenging rate curves show (ICS50): DSS, DSS-E part, DSS-N part, DSS-W part:0.3518,1.012,1.133,1.371mg/mL, Scavenging activity of radical DPPH by the acetate part of extracts from the DSS is the strongest, DSS-E part, DSS-N part, DSS-W part, DSS-P part successively followed.
(3) Use hydrogen peroxide (H2O2) to damage PC12cells,400,450μg/ml concentrations of DSS show cell toxicity. The survival rate of cells was120%、128%、129%、126%、100%、59%、51%(R＜0.05) by different doses of DSS (100,150,200,250,300,350,400μg/ml); The equivalent dose of compound (300μg/ml) parts could antagonize the action of H2O2, the cell survival rate was significantly increased compared with the model group, the difference was statistically significant (P<0.05), but no significant difference between various parts.
Analyze index DSS in blood and tissues of rats by LC/MS/MS technique.
(1) The experimental determination of paeoniflorin, ferulic acid content at different time in the plasma by LC/MS/MS method, which has high sensitivity, rapid, simple, accurate, short analysis time, peak is in5minutes, good reproducibility, and can be used for quantitative paeoniflorin, ferulic acid in rat plasma. But Ligustrazine,23-acetyl Alisol components was not detected, probably because the method sensitivity is not high enough, or it may be these components in rats in vivo into further study is needed to other substances.
Pharmacokinetic parameter show that paeoniflorin in vivo removed rapidly, the half-life is5±1.4h, curve has the double peak phenomenon (Cmax in the first0.5h), second peak occurred at about8-10h (concentration lower than first peak). The dosage and the area under the concentration-time curve of rats, the peak concentration was nonlinear correlation, suggesting paeoniflorin in dynamical behavior of nonlinear dynamics process of rats in vlvo.
(2) Rats were gavaged with DSS, in rat's vivo tissues distribution of high order as follows:
15min in the rat sequence of paeoniflorin concentration order:stomach> liver> lung>intestine> heart> blood> kidney> brain;
35min in the rat sequence of paeoniflorin concentration order:stomach) blood> lung>intestine> kidney> liver> heart> brain;6h in the rat sequence of paeoniflorin concentration order:stomach) blood>intestine> kidney> liver> lung)heart> brain.
15min in rat tissues of ferulic acid concentration order:stomach>intestine> liver> lung> heart> blood)kidney> brain;
35min in rat tissues of ferulic acid concentration order:stomach>intestine) lung>brain> liver) kidney> blood> heart;
6h in rat tissues of ferulic acid concentration order:stomach>intestine> lung> kidney> blood.
Conc Iusions:
Study on the comprehensive use of chemical fingerprint, pharmacodynamics, pharmacokinetics, and joint research pattern that Danggui Shaoyao San mechanism of anti-aging.
Chemical fingerprint:using the HPLC-DAD method of Danggui Shaoyao San and petroleum ether extract, ethyl acetate, n-butanol and water area, part of the main components of paeoniflorin, ferulic acid, ligustrazine, atractylenolide I, the content of ligustilide. Found that the content of each component of Danggui Shaoyao San and in different parts of each level. Established the Danggui Shaoyao San three wavelength (220nm,270nm,320nm) fusion fingerprint, calibration of5control of paeoniflorin, ferulic acid, ligustrazine, atractylenolide I, ligustilide chromatographic peak, the calibration of32characteristic peaks, degree of separation is good, good reproducibility and stability, provide quality guarantee for the pharmacodynamic study.
Pharmacodynamics experiment demonstrated that abnormal and spatial cognitive Danggui Shaoyao San can obviously improve the behavior induced by D-galactose combined with A1Cl3in subacute aging mice, improve the ability of learning and memory; reduce oxidative free radical damage to brain tissue; abnormal tissue caused by improving the pathological factors in experimental animal brain. In vitro antioxidant proved its antioxidant capacity size order: Danggui Shaoyao San, ethyl acetate, n-butanol and water position, petroleum ether fraction. PC12cells in vitro experiments prove that Danggui Shaoyao San and each position can antagonize the action of H2O2, the cell survival rate was significantly increased, but no significant difference between various parts, likely Danggui Shaoyao San showed a protective effect on nerve cells in the overall point of view.
Study on pharmacokinetics showed that rat's plasma paeoniflorin ferulic acid is the main effective components of Danggui Shaoyao San antidementia activity points. Given to rats orally Danggui Shaoyao San, Danggui Shaoyao San can be rapidly distributed in the body most organs, organizations of paeoniflorin and ferulic acid concentration reached the peak of rat5min, Danggui Shaoyao San in rats of paeoniflorin and ferulic acid were the highest content of gastrointestinal tract, the brain with minimum quantity, relative paeoniflorin, ferulic acid elimination rate slow.
Chemical composition and pharmacodynamic study of Danggui Shaoyao San shows that the analysis of Pearson and grey correlation degree:in front of the5known index component of paeoniflorin, ferulic acid, ligustrazine, ligustilide, atractylenolide I and efficacy of highly correlated, scientific combination of pharmacokinetic results in the experiment the mechanical angle5the index components as the pharmacodynamic material bases.
引文
[1]Ferri CP, Prince M, Brayne C, etal, Alzheimers Disease International Global prevalence of dementia:a Delph i consensus study[J]. Lancet,2005, 366(9503):2112-2117.
[2]John C, Morris, Martha Storandt, et al, Mild Cognitive Impairment Represents EarlyStage Disease[J]. Arch Neurol,2001,58:397-405.
[3]Petersen R C, Smith G E, et al.Mild cognitive:clinical characterization and outcome[J]. Arch Neurol,1999,56(3):303-308.
[4]施安国,费艳秋,安富荣,等.老年痴呆症药物治疗进展[J].药学进展,2000,24(6)：338-340.
[5]闫丽莎.盐酸多奈哌齐治疗阿尔茨海默病的临床疗效[J].中华老年心脑血管病杂志,2006,8(2)：829-831.
[6]赵琨,苏红军.老年性痴呆治疗进展[J].华北煤炭医学院学报,2005,7(2)：173-175.
[7]刘艳霞,胡晋红等.加兰他敏作用机制与临床应用[J].世界临床药物.2007,28(7)：420-423.
[8]殷铭俊,陈执中.天然药物抗氧化活性成分研究进展[J].中国民族民间医药杂志,2005,(2)：65-69.
[9]希雨.鼠尾草与早老性痴呆[J].国外医药植物分册,2004,19(6)：242-245.
[10]周鲁,张卫华等.老年痴呆的复方用药规律研究[J].辽宁中医杂志,2005,32(3)：243-244.
[11]陈纪藩.金匮要略[M].北京：人民卫生出版社,2000,1：661-662.
[12]高德义,黄贾生,何宏文.当归芍药散治疗老年性痴呆36例临床研究[J].中国全科医学,2004,7(11)：782-783.
[13]张启春,王秋娟,寇俊萍,等.当归芍药散防治老年期痴呆的物质基础与作用机理研究[J].中国实验方剂学杂志,2005,11,(5)：55-56.
[14]荻野信义.当归芍药散对脑烟碱乙酰胆碱受体合成的刺激作用[J].国外医学.中医中药分册,1988,10(4)：46-48.
[15]马世平.当归芍药散的抗氧化作用研究[J].中药药理与临床,2001,17(3)：123-124.
[16]刘红,曾宇,马世平.不同用量配伍当归芍药散对动物学习记忆功能及脑内NO的影响[J].中国临床药理学与治疗学,2005,10(9)：974-977.
[17]舒斌,马世平,瞿融.当归芍药散对动物学习记忆功能及其单胺递质系统的影
响.[J]江苏中医药,2002,23(6)：34-35.
[18]Huang WH, Song CQ. Chemical and pharmacological advances of the study on Angelica sinensis[J]. China J Chin Mater Med,2001,26(3):147-151.
[19]宋秋月,付迎波,刘江,等.当归的化学成分研究[J].中草药,2011,42(10)：1900-1904.
[20]刘毅,刘素香,龚苏晓,等.当归药材HPLC诣纹图谱及其液相色谱-质谱联用分析[J].药物评价研究,2010,33(4)：259-262.
[21]冯学花,梁肖蕾.当归化学成分与药理作用的研究进展[J].广州化工,2012,44(22)：16-18.
[22]王彦志,石任兵,刘斌.赤芍化学成分的分离与结构鉴定[J].南京中医药大学学报,2006,29(4)：267-269.
[23]Kamiya k, Yoshioka K,Saiki Y, etal.Triterpenoids and flavonoids from paeonia lactiflora[J]. Phytochemistry,1997,44(1):141-144.
[24]何忠梅,孙佳明,张辉,等.固相萃取—高效液相色谱—电喷雾串联质谱法分析芍药和酒芍药的水溶性化学成分[J].分析化学,2009,37(8)：1201-1205.
[25]张晓萍.川芎及其有效成分药理活性研究进展[J].黑龙江医药,2005,18(5)：332-335.
[26]李秋怡,于国平,等.川芎的化学成分及药理研究进展[J].时珍国医国药,2006,17(7)：1298-1299.
[27]张晓川,陈琴华,朱军.白术超临界CO2流体萃取部位脂溶性成分的GC/MS分析[.T].中国药房,2006,17(23)：1835-1837.
[28]林永成.中药白术中一种新的双倍半萜内酯[J].中山大学学报(自然科学版),1995,3(5)：27-30.
[29]胡斌,杨益平,叶阳.茯苓化学成分研究[J].中草药,2006,37(5)：655-657.
[30]Tai T, A kahoriA, Shingu T1 A lano stane triterpenoid from Poria cocos [J].Phy tochemistry,1992,31(7):25482-25491.
[31]朱玉岚,彭国平.泽泻的萜类化学成分研究进展[J].天然产物研究与开发,2006,18(2)：348-350.
[32]Zhou AC, Zhang CF, Zhang M. A New Protostane Triterpenoid from the Rhizome of Alisma orgetale[J]. Chinese Journal of Natural Medicine,2008,2(6):109-111.
[33]陈林霖,王艳慧等.当归芍药散入血成分的分析及药代动力学研究[J],中国天然药物化学,2011,9(5)：363-368.
[34]宋欣,陶春蕾,等.高效液相-二极管阵列法检测当归芍药散中主要化学成分[J],安徽中医学院学报,2012,1(1)：65-66.
[35]贺晓艳,宋秋月等,HPLC法测定当归芍药散中芍药苷、白芍苷、阿魏酸、洋川芎 内酯Ⅰ的含量[J],亚太传统医药,2011,7(7)：36-38.
[36]夏泉,张平,李绍平.当归的药理作用研究进展[J].时珍国医国药,2004,15(3)：164-1661.
[37]赵奎君,钟萌,杨恩来等.当归的炮制对当归补血汤中活性成分的影响[J].中草药,2006,37(12)：1813-1816.
[38]冯学花,梁肖蕾.当归化学成分与药理作用的研究进展[J].广州化工,2012,44(22)：16-18.
[39]冯俊志,赵国平.当归的免疫药理研究进展[J].福建中医药,2003,34(3)：52-55.
[40]夏泉,张平,李绍平.当归的药理作用研究进展[J].时珍国医国药,2004,15(3)：164-1661.
[41]胡慧娟,杭秉茜,王明书.当归的抗炎作用[J].中国中药杂志,2009,16(11)：684-687.
[42]郑敏,王亚平.当归多糖对K562细胞增殖抑制与诱导分化的实验研究[J].中国中西医结合杂志,2002,22(1)：54-57.
[43]黄伟晖,宋纯清.当归的化学和药理学研究进展[J].中国中药杂志,2001,26(3)：147-152.
[44]康旭珍.阿魏酸抗氧化活性研究[J].山西中医学院学报,2005,6(2)：50-52.
[45]Yan J J, Cho JY, K im H S, et al. Protection against beta2 amyloid peptide toxicity in vivo with longterm administration of feru lie acid [J]. Br J Pharmacol,2001,133(1):92-96.
[22]H siehM T, TsaiF H, Lin Y C, et al. Effects of feru lie acid on the impa irment of inhibitory avoidance performance in rats [J]. PlantaMed,2002,68(8): 754-756.
[46]Jiang DaiXun, Chen YiShan, Hou XiaoTao, et al. Influence of Paeonia lactiflora roots extract on Camp-phosphodiesteraseactvity and related anti-inflammatory action[J]. J Ethnophar-macol,2001,137(1):914-920.
[47]吴慧丽,李慧.白芍总苷对溃疡性结肠炎大鼠细胞因子影响的研究[J].中南药学,2010,8(2)：128-131.
[48]]王君萍,韦颖梅,郭梅,等.芍药苷对大鼠脑缺血再灌注损伤细胞[J].中西医结合心脑血管病杂志,2008,6(3)：310-312.
[49]孙蓉,吕丽莉,刘国卿.芍药苷对沙土鼠不全性脑缺血后能量代谢、NO和NOS的影响[J].中国中药杂志,2006,31(10)：832-835.
[50]李文艳,黄山君,王瑞.中药白芍的药理作用和质量控制研究进展[J].药学服务与研究,2012,12(2)：118-120.
[51]陈广斌,陈华萍,吴铁,等.芍药苷对缺氧缺血性脑损伤新生大鼠脑组织丙二醛、 超氧化物歧化酶的影响[J].实用儿科临床杂志,2008,23(16)：1272-1274.
[52]岑得意,陈志武,宋必卫,等.川芎嗪对大鼠脑梗塞的保护作用[J].中国药理学通报,1999,15(5)：464-465.
[53]梁日欣,廖福龙,韩东.川芎嗪预处理对麻醉家兔心肌缺血再灌注损伤的保护作用[J].中药药理与临床,2000,16(2)：11-13.
[54]孙文虹,丁晓飞等,川芎嗪促进大鼠学习记忆作用的研究[J].哈尔滨商业大学学报(自然科学版),2004,20(1)：11-16.
[55]李秋怡,干国平,刘焱文.川芎的化学成分及药理研究进展[J].时珍国医国药,2006,17(7)：1298-1299.
[56]冯星.双白术内酯对A β1-40致痴呆模型大鼠的作用[J].中国药理学通报,2009,25(7)：949-951.
[57]刘洋,廖春梅.双白术内酯对三氯化铝致痴呆模型小鼠的作用研究[J].湖南师范大学学报,2006,3(3)：25-28.
[58]李雯,尹华.白术化学成分的药理作用研究进展[J].海峡药学,2012,24(3)：9-11.
[59]侯安继,陈腾云,彭施萍,等.茯苓多糖抗衰老作用研究[J].中药药理与临床,2004,20(3)：10-11.
[60]张敏,高晓红,等.茯苓的药理作用及研究进展[J].北华大学学报(自然科学版),2008,9(1)：63-66.
[61]高虹,胡应和1用毒蕈胆碱Ml受体激动剂筛选技术发现的泽泻新用途[P]中国专利：CN16159811
[62]易醒,黄丹菲,等.泽泻的研究现状与展望[J].时珍国医国药,2007,18(2)：311-313.
[63]高德义,黄贾生,何宏文.当归芍药散治疗老年性痴呆36例临床研究[J].中国全科医学,2004,7(11)：782-78.
[64]林志宏,严永清,朱丹妮,等.当归芍药散对β-淀粉样蛋白致PC12细胞损伤的保护作用[J].中国药科大学学报,2005,36(4)：346-349.
[65]田荣波,李铁浪.当归芍药散抗衰老的研究进展[J].中医药导报,2006,12(5)：102-107.
[66]阿依努尔,任红莉,蔡轶,等.当归芍药散抗痛经作用的药效学研究[J].西北药学杂志,2009,4(2)：118-119.
[67]陶正恒.当归芍药散的研究进展[J].中药材,2000,1(23)：58-61.
[68]舒斌,马世平,瞿融.当归芍药散对动物学习记忆功能及其单胺递质系统的影响[J].江苏中医药,2002,23(6)：34-38.
[69]田荣波,李铁浪.当归芍药散抗衰老的研究进展[R].中医药导报,2006,12(5)：102-107.
[70]荻野信义,等.当归芍药散对脑烟碱乙酰胆碱受体合成的刺激作用[J].国外医学·中医中药分册,1988,10(4)：46-49.
[71]阎艳丽,王鑫国,宋晓宇.当归芍药散对高脂血症家兔脂代谢及血液流变学的影响[J].辽宁中医杂志,2005,32(2)：170-171.
[72]何宏文,姚志彬.当归芍药散对老年小鼠SOD活性、MDA含量的影响[J].中国民政医学杂志,1998,10(5)：297-298.
[73]侯集瑞,孙晓波,徐惠渡,温富春,周继胡,丁涛.当归芍药散药理与临床研究进展[J].中国中医药信息杂志,2002,9(9)：26-28.
[74]寇俊萍,华敏,严永清.当归芍药散对小鼠免疫功能的影响[J].中国现代应用药学,2003,20(3)：171-174.
[75]陶正恒,等.当归芍药散的研究进展[J].中药材,2000,1(23)：58-61.
[76]藤原道弘,当归芍药散对大鼠空间识别和被动回避反射障碍的影响[J].和汉医学会志,1989;6(3)：234-235.
[77]尾崎哲.对当归芍药散趋精神作用的认识[J].新药与临床,1992,41(3)：220-222.
[78]水岛宣昭.当归芍药散对老年痴呆的作用[J].和汉医药学会志,1989,6(3)：456-458.
[79]童珊珊,余江南,刘文英.体内药物分析中的样品预处理技术[J].药学进展,2001,25(1)：24-26.
[80]周莹,杜武勋,朱明丹,等.中药复方药代动力学研究方法进展[J].辽宁中医药大学学报,2010,12(5)：143-144.
[81]李文兰,程斌,季宇彬.金匮肾气丸血清样品预处理方法的研究[J].黑龙江大学自然科学学报,2007,24(6)：792-794.
[82]张莉,黄熙,王骊丽.中药药动学血样预处理方法回顾与展望[J].中草药,2000,31(10)：792-794.
[83]Poole C F, New trends in solid phase extraction[J]. Trends Anal.Chem. 2003,22(6):362-373.
[84]Zhang ZY, Poerschmann J, Panliszyn J, et al. Directsolid phase microextraraction of complex aqueous samlples with hollow fibre membrane p rotection [J].Anal Commu,1996,33(7):219-222.
[85]Gorecki T, Mindrup R, Pawliszyn. Pesticides by solid-phase micmestraction results of round robin test[J].J. Analyst,1996,121(10):1381-1386.
[86]Chester TL, Pink ston JD, Raynie DE. Supercritical fluid chromatography and extraction [J]. Anal Chem,1998,70 (12):301-319.
[87]MeloLP, Nogueira M, LanAs F M, et al. Polydimethylsiloxane/polypyrrolestir bar sorptive extraction and liquid chromatography (SBSE/LC-UV) analysis of antidepressants in plasma samples[J]. Analytica Chimica Acta.2009,
633(1):57-64.
[88]Safaralie A, Fatemi S, Sefidkon F. Essential oil composition of Valeriana officinalis L. roots cultivated in Iran Comparative analysis between supercritical CO2 extraction and hydrodistillation[J]. Journal of Chromatography A,2008,1180(1-2):159-164.
[89]李日红,微透析技术在植物活体取样中的应用研究[M].《辽宁师范大学》2010年硕士论文.
[90]Rezaei B, Malakpour S, Majidi N. Solid-phase molecularly imprintedpre-concentration and spectrophotometric determination of isoxicam inpharmaceuticals and human serum[J]. Talanta.2009,78(2):418-423.
[91]秦彩玲,刘婷,张毅,等.参麦注射液抗大鼠内毒素休克药效学和药动学探讨[J].中国实验方剂学杂志,2000,6(3)：22-24.
[92]肖凤霞,周莉玲,李锐.药物累积法测定四逆汤制剂的药动学参数[J].时珍国医国药,2006,17(2)：228-229.
[93]杨志福,乔逸,何仲贵,等.红花提取物在急性血瘀大鼠体内的药物动力学研究[J].陕西中医,2009,30(9)：1222-1224.
[94]姜少灏,康丽娟,蒋晔,等.龙胆及其复方中龙胆苦苷在大鼠体内的药动学研究[J].中国药学杂志,2005,40(3)：212-215.
[95]沈群,罗佳波.重视中药复方药动学研究的特殊性[P].中国医药报/2005,1月13日.
[96]王广基,中药药代动力学研究的新探索[M].第十届全国药物和化学异物代谢学术会议暨第三届ISSX/CSSX联合学术会议：5-6.
[97]罗焕敏,肖飞,D-半乳糖和三氯化铝诱导小鼠产生类阿尔茨海默病变[J].中国药理学与毒理学杂志.2004,18(1)：22-26.
[98]叶红梅,钟春燕,吕俊华.荔枝核提取物改善D-半乳糖诱致小鼠学习记忆障碍与体内抗氧化作用的关系[J].海峡药学,2012,24(1)：25-28.
[99]苏婧,贺海波,石孟琼,等.火麻仁油对D-半乳糖致衰老学习小鼠学习记忆的保护作用研究[J].中国临床药理学与治疗学,2011,16(12)：1332-1339.
[100]秦海强,李亚,田苏平,等.β-淀粉样蛋白及D-半乳糖对大鼠学习记忆和胆碱酯酶活性的影响[J].南京医科大学学报,2000(03)：43-45.
[101]董自波,洪敏,樊宏伟,等.当归HPLC指纹图谱研究[J].中国实验方剂学杂志,2004,10(3)：1-4.
[102]李越峰,严兴科,沈菲,等.白芍HPLC指纹图谱相似度的分析研究[J].中成药,2008,30(7)：942-946.
[103]宋金春,胡传芹,曾俊芬,等.川芎RP-HPLC指纹图谱的建立及其质量研究[J].中国药师,2006,9(4)：297-302.
[104]叶燕,程雪梅,俞桂新,等.白术药材HPLC指纹图谱研究[J].江苏中医药,2009,41(4)：59-61.
[105]钟凌云,段启,龚千锋.反相高效液相色谱法测定茯苓中茯苓酸的含量[J].中国中医药信息杂志,2005,12(12)：2531-2531.
[106]吴士杰,涂正伟,安海洋,等.泽泻高效液相色谱指纹图谱研究[J].中国药业,2011,20(6)：19-23.
[107]何建雄,魏刚,黄月纯.广蕾香GC-MS特征指纹图谱数字化信息的应用及其GC验证[J].中国中药杂志,2008,33(03)：316-319.
[108]安耀荣,朱向东,王燕.抗衰益智胶囊对实验性老年痴呆病大鼠脑组织中SOD、CAT活性及MDA含量的影响[J].中医研究,2009.6：26-27
[109]XU Jiang-Ping, WU Hang-Yu, LI Lin Effects of compound Yi-Zhi on D-galactose-induced learningand memory deficits in mice Journal of Neuropathology and Experimental Neurology.2003 Feb; 8(1):31-34
[110]Li WB, Wei F, Fan M, et al. Mimetic brain aging effect induced by D-galactose in mice[J]. Chin J Pharmacol Toxicol,1995,9:93-95.
[111]宋琳.地黄引子改善痴呆大鼠学习记忆及其防治AD作用机理的实验研究.博士论文,黑龙江中医药大学,2005.
[112]李美珍,孙必强,曾姣飞,等.左归丸对老年性痴呆模型大鼠SOD活性和MDA含量的影响[J].中华中医药学期刊,2010,12(3)：32-35.
[113]朱亚珍,朱宏光D-半乳糖致衰老动物模型的建立及其检法[J].复旦学报.2007.3(34),617-618
[114]彭颖,吴晓波.中药抗阿尔茨海默病的研究进展[J].世界医学—中医药现代化.2005,75)：63-67.
[115]张均田.神经药理学研究进展[M].人民卫生出版社,2002,19-29.
[116]刘江红.p-淀粉样蛋白致神经细胞损伤和保护因素拮抗损伤的机制及信号转导通路初步研究.博士学位论文,中国协和医科大学,2004.
[117]冯改利,宋小妹,邓习中,等.DPPH法筛选大血藤抗氧化活性有效部位[J]陕西中医学院学报,2011,28(6)：1233-1235.
[118]王晶,李丽,刘春明,等.不同红药提取物中总酚的测定及抗氧化活性的DPPH法评价研究[J].辽宁中医杂志,2011,38(3)：513-515.
[119]Hu F1,Lu RL, Huang B, et al. Free radical scavenging activity of extracts Prepared from fresh leaves of selected Chinese medicinal plants[J]. Fitoterapia,2004,75:14-23.
[120]于德红,魏朝良,包永明,等.阿尔茨海默病与氧化应激[J].生物医学工程学杂志,2006,23(5)：1142-1144.
[121]Kaminsky YG, Kosenko EA. Effects of amyloid-beta peptides onhydrogen peroxide-metabolizing enzymes in rat brain in vivo[J].Free Radic Res,2008,42 (6):564-573.
[122]刘丽萍,阿尔茨海默病病理学研究进展[J].中国临床康复,2003,7(5)：785-786.
[123]Du XJ, Zhang JS, Liu YF, etal. The antioxidant activity of various extracts from Tremella aurantialba fruiting bodies and their protective effects on PC,2 cells injured by oxidation[J]. Acta Agric Shanghai,2010,26(2):49-52.
[124]沈群罗佳波,重视中药复方药动学研究的特殊性[P].中国医药报/2005,1月13日.
[125]刘建勋,林成仁,前列安栓在体内的吸收与分布[J].新中医,2000,32(4)：59-61.
[2]John C, Morris, Martha Storandt, et al, Mild Cognitive Impairment Represents EarlyStage Disease[J]. Arch Neurol,2001,58:397-405.
[3]Petersen R C, Smith G E, et al.Mild cognitive:clinical characterization and outcome[J]. Arch Neurol,1999,56(3):303-308.
[4]施安国,费艳秋,安富荣,等.老年痴呆症药物治疗进展[J].药学进展,2000,24(6)：338-340.
[5]闫丽莎.盐酸多奈哌齐治疗阿尔茨海默病的临床疗效[J].中华老年心脑血管病杂志,2006,8(2)：829-831.
[6]赵琨,苏红军.老年性痴呆治疗进展[J].华北煤炭医学院学报,2005,7(2)：173-175.
[7]刘艳霞,胡晋红等.加兰他敏作用机制与临床应用[J].世界临床药物.2007,28(7)：420-423.
[8]殷铭俊,陈执中.天然药物抗氧化活性成分研究进展[J].中国民族民间医药杂志,2005,(2)：65-69.
[9]希雨.鼠尾草与早老性痴呆[J].国外医药植物分册,2004,19(6)：242-245.
[10]周鲁,张卫华等.老年痴呆的复方用药规律研究[J].辽宁中医杂志,2005,32(3)：243-244.
[11]陈纪藩.金匮要略[M].北京：人民卫生出版社,2000,1：661-662.
[12]高德义,黄贾生,何宏文.当归芍药散治疗老年性痴呆36例临床研究[J].中国全科医学,2004,7(11)：782-783.
[13]张启春,王秋娟,寇俊萍,等.当归芍药散防治老年期痴呆的物质基础与作用机理研究[J].中国实验方剂学杂志,2005,11,(5)：55-56.
[14]荻野信义.当归芍药散对脑烟碱乙酰胆碱受体合成的刺激作用[J].国外医学.中医中药分册,1988,10(4)：46-48.
[15]马世平.当归芍药散的抗氧化作用研究[J].中药药理与临床,2001,17(3)：123-124.
[16]刘红,曾宇,马世平.不同用量配伍当归芍药散对动物学习记忆功能及脑内NO的影响[J].中国临床药理学与治疗学,2005,10(9)：974-977.
[17]舒斌,马世平,瞿融.当归芍药散对动物学习记忆功能及其单胺递质系统的影
响.[J]江苏中医药,2002,23(6)：34-35.
[18]Huang WH, Song CQ. Chemical and pharmacological advances of the study on Angelica sinensis[J]. China J Chin Mater Med,2001,26(3):147-151.
[19]宋秋月,付迎波,刘江,等.当归的化学成分研究[J].中草药,2011,42(10)：1900-1904.
[20]刘毅,刘素香,龚苏晓,等.当归药材HPLC诣纹图谱及其液相色谱-质谱联用分析[J].药物评价研究,2010,33(4)：259-262.
[21]冯学花,梁肖蕾.当归化学成分与药理作用的研究进展[J].广州化工,2012,44(22)：16-18.
[22]王彦志,石任兵,刘斌.赤芍化学成分的分离与结构鉴定[J].南京中医药大学学报,2006,29(4)：267-269.
[23]Kamiya k, Yoshioka K,Saiki Y, etal.Triterpenoids and flavonoids from paeonia lactiflora[J]. Phytochemistry,1997,44(1):141-144.
[24]何忠梅,孙佳明,张辉,等.固相萃取—高效液相色谱—电喷雾串联质谱法分析芍药和酒芍药的水溶性化学成分[J].分析化学,2009,37(8)：1201-1205.
[25]张晓萍.川芎及其有效成分药理活性研究进展[J].黑龙江医药,2005,18(5)：332-335.
[26]李秋怡,于国平,等.川芎的化学成分及药理研究进展[J].时珍国医国药,2006,17(7)：1298-1299.
[27]张晓川,陈琴华,朱军.白术超临界CO2流体萃取部位脂溶性成分的GC/MS分析[.T].中国药房,2006,17(23)：1835-1837.
[28]林永成.中药白术中一种新的双倍半萜内酯[J].中山大学学报(自然科学版),1995,3(5)：27-30.
[29]胡斌,杨益平,叶阳.茯苓化学成分研究[J].中草药,2006,37(5)：655-657.
[30]Tai T, A kahoriA, Shingu T1 A lano stane triterpenoid from Poria cocos [J].Phy tochemistry,1992,31(7):25482-25491.
[31]朱玉岚,彭国平.泽泻的萜类化学成分研究进展[J].天然产物研究与开发,2006,18(2)：348-350.
[32]Zhou AC, Zhang CF, Zhang M. A New Protostane Triterpenoid from the Rhizome of Alisma orgetale[J]. Chinese Journal of Natural Medicine,2008,2(6):109-111.
[33]陈林霖,王艳慧等.当归芍药散入血成分的分析及药代动力学研究[J],中国天然药物化学,2011,9(5)：363-368.
[34]宋欣,陶春蕾,等.高效液相-二极管阵列法检测当归芍药散中主要化学成分[J],安徽中医学院学报,2012,1(1)：65-66.
[35]贺晓艳,宋秋月等,HPLC法测定当归芍药散中芍药苷、白芍苷、阿魏酸、洋川芎 内酯Ⅰ的含量[J],亚太传统医药,2011,7(7)：36-38.
[36]夏泉,张平,李绍平.当归的药理作用研究进展[J].时珍国医国药,2004,15(3)：164-1661.
[37]赵奎君,钟萌,杨恩来等.当归的炮制对当归补血汤中活性成分的影响[J].中草药,2006,37(12)：1813-1816.
[38]冯学花,梁肖蕾.当归化学成分与药理作用的研究进展[J].广州化工,2012,44(22)：16-18.
[39]冯俊志,赵国平.当归的免疫药理研究进展[J].福建中医药,2003,34(3)：52-55.
[40]夏泉,张平,李绍平.当归的药理作用研究进展[J].时珍国医国药,2004,15(3)：164-1661.
[41]胡慧娟,杭秉茜,王明书.当归的抗炎作用[J].中国中药杂志,2009,16(11)：684-687.
[42]郑敏,王亚平.当归多糖对K562细胞增殖抑制与诱导分化的实验研究[J].中国中西医结合杂志,2002,22(1)：54-57.
[43]黄伟晖,宋纯清.当归的化学和药理学研究进展[J].中国中药杂志,2001,26(3)：147-152.
[44]康旭珍.阿魏酸抗氧化活性研究[J].山西中医学院学报,2005,6(2)：50-52.
[45]Yan J J, Cho JY, K im H S, et al. Protection against beta2 amyloid peptide toxicity in vivo with longterm administration of feru lie acid [J]. Br J Pharmacol,2001,133(1):92-96.
[22]H siehM T, TsaiF H, Lin Y C, et al. Effects of feru lie acid on the impa irment of inhibitory avoidance performance in rats [J]. PlantaMed,2002,68(8): 754-756.
[46]Jiang DaiXun, Chen YiShan, Hou XiaoTao, et al. Influence of Paeonia lactiflora roots extract on Camp-phosphodiesteraseactvity and related anti-inflammatory action[J]. J Ethnophar-macol,2001,137(1):914-920.
[47]吴慧丽,李慧.白芍总苷对溃疡性结肠炎大鼠细胞因子影响的研究[J].中南药学,2010,8(2)：128-131.
[48]]王君萍,韦颖梅,郭梅,等.芍药苷对大鼠脑缺血再灌注损伤细胞[J].中西医结合心脑血管病杂志,2008,6(3)：310-312.
[49]孙蓉,吕丽莉,刘国卿.芍药苷对沙土鼠不全性脑缺血后能量代谢、NO和NOS的影响[J].中国中药杂志,2006,31(10)：832-835.
[50]李文艳,黄山君,王瑞.中药白芍的药理作用和质量控制研究进展[J].药学服务与研究,2012,12(2)：118-120.
[51]陈广斌,陈华萍,吴铁,等.芍药苷对缺氧缺血性脑损伤新生大鼠脑组织丙二醛、 超氧化物歧化酶的影响[J].实用儿科临床杂志,2008,23(16)：1272-1274.
[52]岑得意,陈志武,宋必卫,等.川芎嗪对大鼠脑梗塞的保护作用[J].中国药理学通报,1999,15(5)：464-465.
[53]梁日欣,廖福龙,韩东.川芎嗪预处理对麻醉家兔心肌缺血再灌注损伤的保护作用[J].中药药理与临床,2000,16(2)：11-13.
[54]孙文虹,丁晓飞等,川芎嗪促进大鼠学习记忆作用的研究[J].哈尔滨商业大学学报(自然科学版),2004,20(1)：11-16.
[55]李秋怡,干国平,刘焱文.川芎的化学成分及药理研究进展[J].时珍国医国药,2006,17(7)：1298-1299.
[56]冯星.双白术内酯对A β1-40致痴呆模型大鼠的作用[J].中国药理学通报,2009,25(7)：949-951.
[57]刘洋,廖春梅.双白术内酯对三氯化铝致痴呆模型小鼠的作用研究[J].湖南师范大学学报,2006,3(3)：25-28.
[58]李雯,尹华.白术化学成分的药理作用研究进展[J].海峡药学,2012,24(3)：9-11.
[59]侯安继,陈腾云,彭施萍,等.茯苓多糖抗衰老作用研究[J].中药药理与临床,2004,20(3)：10-11.
[60]张敏,高晓红,等.茯苓的药理作用及研究进展[J].北华大学学报(自然科学版),2008,9(1)：63-66.
[61]高虹,胡应和1用毒蕈胆碱Ml受体激动剂筛选技术发现的泽泻新用途[P]中国专利：CN16159811
[62]易醒,黄丹菲,等.泽泻的研究现状与展望[J].时珍国医国药,2007,18(2)：311-313.
[63]高德义,黄贾生,何宏文.当归芍药散治疗老年性痴呆36例临床研究[J].中国全科医学,2004,7(11)：782-78.
[64]林志宏,严永清,朱丹妮,等.当归芍药散对β-淀粉样蛋白致PC12细胞损伤的保护作用[J].中国药科大学学报,2005,36(4)：346-349.
[65]田荣波,李铁浪.当归芍药散抗衰老的研究进展[J].中医药导报,2006,12(5)：102-107.
[66]阿依努尔,任红莉,蔡轶,等.当归芍药散抗痛经作用的药效学研究[J].西北药学杂志,2009,4(2)：118-119.
[67]陶正恒.当归芍药散的研究进展[J].中药材,2000,1(23)：58-61.
[68]舒斌,马世平,瞿融.当归芍药散对动物学习记忆功能及其单胺递质系统的影响[J].江苏中医药,2002,23(6)：34-38.
[69]田荣波,李铁浪.当归芍药散抗衰老的研究进展[R].中医药导报,2006,12(5)：102-107.
[70]荻野信义,等.当归芍药散对脑烟碱乙酰胆碱受体合成的刺激作用[J].国外医学·中医中药分册,1988,10(4)：46-49.
[71]阎艳丽,王鑫国,宋晓宇.当归芍药散对高脂血症家兔脂代谢及血液流变学的影响[J].辽宁中医杂志,2005,32(2)：170-171.
[72]何宏文,姚志彬.当归芍药散对老年小鼠SOD活性、MDA含量的影响[J].中国民政医学杂志,1998,10(5)：297-298.
[73]侯集瑞,孙晓波,徐惠渡,温富春,周继胡,丁涛.当归芍药散药理与临床研究进展[J].中国中医药信息杂志,2002,9(9)：26-28.
[74]寇俊萍,华敏,严永清.当归芍药散对小鼠免疫功能的影响[J].中国现代应用药学,2003,20(3)：171-174.
[75]陶正恒,等.当归芍药散的研究进展[J].中药材,2000,1(23)：58-61.
[76]藤原道弘,当归芍药散对大鼠空间识别和被动回避反射障碍的影响[J].和汉医学会志,1989;6(3)：234-235.
[77]尾崎哲.对当归芍药散趋精神作用的认识[J].新药与临床,1992,41(3)：220-222.
[78]水岛宣昭.当归芍药散对老年痴呆的作用[J].和汉医药学会志,1989,6(3)：456-458.
[79]童珊珊,余江南,刘文英.体内药物分析中的样品预处理技术[J].药学进展,2001,25(1)：24-26.
[80]周莹,杜武勋,朱明丹,等.中药复方药代动力学研究方法进展[J].辽宁中医药大学学报,2010,12(5)：143-144.
[81]李文兰,程斌,季宇彬.金匮肾气丸血清样品预处理方法的研究[J].黑龙江大学自然科学学报,2007,24(6)：792-794.
[82]张莉,黄熙,王骊丽.中药药动学血样预处理方法回顾与展望[J].中草药,2000,31(10)：792-794.
[83]Poole C F, New trends in solid phase extraction[J]. Trends Anal.Chem. 2003,22(6):362-373.
[84]Zhang ZY, Poerschmann J, Panliszyn J, et al. Directsolid phase microextraraction of complex aqueous samlples with hollow fibre membrane p rotection [J].Anal Commu,1996,33(7):219-222.
[85]Gorecki T, Mindrup R, Pawliszyn. Pesticides by solid-phase micmestraction results of round robin test[J].J. Analyst,1996,121(10):1381-1386.
[86]Chester TL, Pink ston JD, Raynie DE. Supercritical fluid chromatography and extraction [J]. Anal Chem,1998,70 (12):301-319.
[87]MeloLP, Nogueira M, LanAs F M, et al. Polydimethylsiloxane/polypyrrolestir bar sorptive extraction and liquid chromatography (SBSE/LC-UV) analysis of antidepressants in plasma samples[J]. Analytica Chimica Acta.2009,
633(1):57-64.
[88]Safaralie A, Fatemi S, Sefidkon F. Essential oil composition of Valeriana officinalis L. roots cultivated in Iran Comparative analysis between supercritical CO2 extraction and hydrodistillation[J]. Journal of Chromatography A,2008,1180(1-2):159-164.
[89]李日红,微透析技术在植物活体取样中的应用研究[M].《辽宁师范大学》2010年硕士论文.
[90]Rezaei B, Malakpour S, Majidi N. Solid-phase molecularly imprintedpre-concentration and spectrophotometric determination of isoxicam inpharmaceuticals and human serum[J]. Talanta.2009,78(2):418-423.
[91]秦彩玲,刘婷,张毅,等.参麦注射液抗大鼠内毒素休克药效学和药动学探讨[J].中国实验方剂学杂志,2000,6(3)：22-24.
[92]肖凤霞,周莉玲,李锐.药物累积法测定四逆汤制剂的药动学参数[J].时珍国医国药,2006,17(2)：228-229.
[93]杨志福,乔逸,何仲贵,等.红花提取物在急性血瘀大鼠体内的药物动力学研究[J].陕西中医,2009,30(9)：1222-1224.
[94]姜少灏,康丽娟,蒋晔,等.龙胆及其复方中龙胆苦苷在大鼠体内的药动学研究[J].中国药学杂志,2005,40(3)：212-215.
[95]沈群,罗佳波.重视中药复方药动学研究的特殊性[P].中国医药报/2005,1月13日.
[96]王广基,中药药代动力学研究的新探索[M].第十届全国药物和化学异物代谢学术会议暨第三届ISSX/CSSX联合学术会议：5-6.
[97]罗焕敏,肖飞,D-半乳糖和三氯化铝诱导小鼠产生类阿尔茨海默病变[J].中国药理学与毒理学杂志.2004,18(1)：22-26.
[98]叶红梅,钟春燕,吕俊华.荔枝核提取物改善D-半乳糖诱致小鼠学习记忆障碍与体内抗氧化作用的关系[J].海峡药学,2012,24(1)：25-28.
[99]苏婧,贺海波,石孟琼,等.火麻仁油对D-半乳糖致衰老学习小鼠学习记忆的保护作用研究[J].中国临床药理学与治疗学,2011,16(12)：1332-1339.
[100]秦海强,李亚,田苏平,等.β-淀粉样蛋白及D-半乳糖对大鼠学习记忆和胆碱酯酶活性的影响[J].南京医科大学学报,2000(03)：43-45.
[101]董自波,洪敏,樊宏伟,等.当归HPLC指纹图谱研究[J].中国实验方剂学杂志,2004,10(3)：1-4.
[102]李越峰,严兴科,沈菲,等.白芍HPLC指纹图谱相似度的分析研究[J].中成药,2008,30(7)：942-946.
[103]宋金春,胡传芹,曾俊芬,等.川芎RP-HPLC指纹图谱的建立及其质量研究[J].中国药师,2006,9(4)：297-302.
[104]叶燕,程雪梅,俞桂新,等.白术药材HPLC指纹图谱研究[J].江苏中医药,2009,41(4)：59-61.
[105]钟凌云,段启,龚千锋.反相高效液相色谱法测定茯苓中茯苓酸的含量[J].中国中医药信息杂志,2005,12(12)：2531-2531.
[106]吴士杰,涂正伟,安海洋,等.泽泻高效液相色谱指纹图谱研究[J].中国药业,2011,20(6)：19-23.
[107]何建雄,魏刚,黄月纯.广蕾香GC-MS特征指纹图谱数字化信息的应用及其GC验证[J].中国中药杂志,2008,33(03)：316-319.
[108]安耀荣,朱向东,王燕.抗衰益智胶囊对实验性老年痴呆病大鼠脑组织中SOD、CAT活性及MDA含量的影响[J].中医研究,2009.6：26-27
[109]XU Jiang-Ping, WU Hang-Yu, LI Lin Effects of compound Yi-Zhi on D-galactose-induced learningand memory deficits in mice Journal of Neuropathology and Experimental Neurology.2003 Feb; 8(1):31-34
[110]Li WB, Wei F, Fan M, et al. Mimetic brain aging effect induced by D-galactose in mice[J]. Chin J Pharmacol Toxicol,1995,9:93-95.
[111]宋琳.地黄引子改善痴呆大鼠学习记忆及其防治AD作用机理的实验研究.博士论文,黑龙江中医药大学,2005.
[112]李美珍,孙必强,曾姣飞,等.左归丸对老年性痴呆模型大鼠SOD活性和MDA含量的影响[J].中华中医药学期刊,2010,12(3)：32-35.
[113]朱亚珍,朱宏光D-半乳糖致衰老动物模型的建立及其检法[J].复旦学报.2007.3(34),617-618
[114]彭颖,吴晓波.中药抗阿尔茨海默病的研究进展[J].世界医学—中医药现代化.2005,75)：63-67.
[115]张均田.神经药理学研究进展[M].人民卫生出版社,2002,19-29.
[116]刘江红.p-淀粉样蛋白致神经细胞损伤和保护因素拮抗损伤的机制及信号转导通路初步研究.博士学位论文,中国协和医科大学,2004.
[117]冯改利,宋小妹,邓习中,等.DPPH法筛选大血藤抗氧化活性有效部位[J]陕西中医学院学报,2011,28(6)：1233-1235.
[118]王晶,李丽,刘春明,等.不同红药提取物中总酚的测定及抗氧化活性的DPPH法评价研究[J].辽宁中医杂志,2011,38(3)：513-515.
[119]Hu F1,Lu RL, Huang B, et al. Free radical scavenging activity of extracts Prepared from fresh leaves of selected Chinese medicinal plants[J]. Fitoterapia,2004,75:14-23.
[120]于德红,魏朝良,包永明,等.阿尔茨海默病与氧化应激[J].生物医学工程学杂志,2006,23(5)：1142-1144.
[121]Kaminsky YG, Kosenko EA. Effects of amyloid-beta peptides onhydrogen peroxide-metabolizing enzymes in rat brain in vivo[J].Free Radic Res,2008,42 (6):564-573.
[122]刘丽萍,阿尔茨海默病病理学研究进展[J].中国临床康复,2003,7(5)：785-786.
[123]Du XJ, Zhang JS, Liu YF, etal. The antioxidant activity of various extracts from Tremella aurantialba fruiting bodies and their protective effects on PC,2 cells injured by oxidation[J]. Acta Agric Shanghai,2010,26(2):49-52.
[124]沈群罗佳波,重视中药复方药动学研究的特殊性[P].中国医药报/2005,1月13日.
[125]刘建勋,林成仁,前列安栓在体内的吸收与分布[J].新中医,2000,32(4)：59-61.
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