CN115054671A - 通脑益智的中药组合物及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种通脑益智的中药组合物及其制备方法和应用,该中药组合物围绕痴呆“毒损脑络”的病理机制,以大黄、水蛭共为君药,黄芪、郁金、川芎共为臣药,枸杞子、益智仁、仙鹤草、石菖蒲共为佐使药。全方以补肾益精、活血通瘀、化痰泄毒为治疗方法,达到改善血管性认知障碍,缓解血管性认知障碍病理学产物聚集的作用,通过各药材的合理配比,协同作用,对治疗血管性认知障碍有明确的治疗作用,同时对缺血后脑神经元的损伤具有保护作用;还通过作用于肠道菌群,降低肠道通透性、减轻肠道炎症,改善认知障碍患者的临床症状。
Description
技术领域
本发明涉及一种通脑益智的中药组合物及其制备方法和应用,尤其涉及一种能够有效治疗血管性认知障碍的中药组合物及其制备方法和应用。
背景技术
血管性认知障碍(vascular cognitive impairment,VCI)是脑血管疾病及其危险因素导致的临床卒中或亚临床脑血管病相关的认知障碍综合征,涵盖了血管性痴呆(vascular dementia,VaD)、伴血管病变的阿尔茨海默病(Alzheimer′s disease,AD)和非痴呆型血管性认知损害(vascular cognitive impairment no dementia,VCIND)等。VCIND是目前公认的唯一可防可控的轻度认知损害,若任由病情发展,VCIND将会很快转变为不可逆的血管性痴呆阶段,届时采取治疗的效果也将会大大降低。因此要早期识别出VCIND患者,采用综合治疗延缓其疾病进程。
VCI的预防主要集中在生活方式干预和预防控制高血压、肥胖、吸烟和糖尿病等血管风险因素。改变不良生活方式对VCI有预防作用,具体措施包括增加认知训练(读书、看报、社交等)、规律的体育锻炼、戒烟戒酒、地中海饮食结构等。目前根据指南推荐常用的改善VCI认知的药物有胆碱酯酶抑制剂、N-甲基-D-天冬氨酸受体拮抗剂、钙离子通道拮抗剂等,然而,上述治疗药物只能在有限的时间里控制痴呆症的症状,不能预防或逆转疾病的进展。因此,如何预防血管性认知障碍的发生、延缓病情进展、缓解痴呆症状、甚至逆转痴呆病程,开发更多有效的治疗药物以更好地保护VCI患者的健康状况是一个严重的社会健康问题。
发明内容
发明目的:针对现有药物存在的不能有效预防或逆转血管性认知障碍进展等问题,本发明旨在提供一种具有补肾益精、活血通瘀、化痰泄毒功效,可有效预防或/和治疗血管性认知障碍的中药组合物及其制备方法和应用。
技术方案:作为本发明涉及的第一方面,本发明的中药组合物按重量计,包含益智仁10~20份、水蛭3~10份、石菖蒲10~25份、枸杞10~20份、炙黄芪10~30份、仙鹤草12~30份、郁金5~15份、川芎5~20份和制大黄5~10份。
《金匮要略心典》曰:“邪气蕴结不解谓之毒。”所谓“毒”,有内外之分,“外毒”指虚邪贼风等不正之邪,“内毒”指脏腑虚损,功能失调,气血津液运行失常,体内痰浊、瘀血等代谢产物不能及时排出,郁蒸腐化,化热成毒,秽气浊毒内生,产生“毒邪”。本发明认为“毒损脑络”是痴呆的主要病机,所谓“毒损脑络”,即以上虚邪贼风等不正之邪以及痰毒、瘀毒、热毒留滞脑络,损伤脑络,或堵塞脑络,致脑络失养,髓海空虚,引起痴呆,故“毒损脑络”是痴呆的核心病机,治疗当予泄毒开窍法因势利导,使毒有去处,则脑神醒转,呆病可缓。
本方中大黄泻下攻逐之力强,清热解毒力专,可消除一切水食痰瘀等有形积滞。生大黄性味苦寒,易于败坏胃气,而大黄制用后不仅能缓生大黄之苦寒峻烈,还能增强活血化瘀之功。大黄中的有效成分芦荟大黄素能够改善痴呆小鼠的记忆功能,且可保护缺血再灌注小鼠的记忆能力。大黄中的有效成分通过调节细胞因子水平,减少氧自由基产生,保护缺血性脑损伤后的神经细胞,可减轻炎症、细胞毒性、凝血等反应。本方针对瘀血阻滞脑络日久,病灶多具有胶着痼结之特点,寻常草木之品恐难有良效,还利用有搜筋剔络之功的虫类药,祛除久踞脑府之瘀血以通利血道。水蛭作为虫类药,味咸性平,能下能软,可破瘀血、通筋络。水蛭水煎剂有较强的抗凝血作用,可延长纤维蛋白的凝聚时间,还可降血脂,消退动脉粥样硬化斑块以及影响血液流变学等多重功效。大黄和水蛭合用,破瘀力强而不伤正气,使脑窍通,脑神转,二者共为君药。
《黄帝内经》曰:“人之所有者,血与气耳。”气血是生命的根本。气虚则人体之津液运行不畅,留滞筋脉,久而成瘀。本方中黄芪大补元气,兼有安神益智、升阳固表之功,用于治疗一切疾病因元气虚而出现的体虚、倦怠。川芎辛温,可“上行巅顶,下达血海,旁通四肢,外彻皮毛,为血中之气药”,适宜瘀血阻滞之各种病症。川芎根茎提取物川芎嗪可保护损伤的海马神经元,同时可增强中枢神经系统中乙酰胆碱的活性,改善脑部循环,从而达到保护脑神经的作用。郁金功效活血止痛,行气解郁,具有调节神经递质、抑制神经炎症、保护神经元、调节神经内分泌、抗血栓等作用。气行血则行,血足气则盛。血脉畅通会改善大脑缺血缺氧状态,醒脑提神,维持人体生理平衡。故黄芪、郁金、川芎合用,鼓舞气血,通利血脉,共为臣药。
枸杞子、益智仁滋肾填精生髓,温阳益肾;石菖蒲祛痰开窍,安神定志;仙鹤草解毒、补虚、抗疲劳。仙鹤草的鞣质分子可清除各种氧自由基;益智仁的乙醇提取物能够抑制神经细胞tau蛋白的磷酸化,从而保护大脑神经元;益智仁水提取物还可有效改善实验动物的学习、记忆能力;石菖蒲及其成分α-细辛醚能够明显降低大鼠海马脑组织中MDA含量,抑制SOD、NOS活性及nNOS蛋白表达,从而改善疲劳运动大鼠学习记忆能力。以上四味合用,补肾填精益智,共为佐使药。
上述诸药合用,针对痴呆“毒损脑络”的病理机制,以大黄及水蛭共为君药,实现活血祛瘀、泄毒通络的功效。石菖蒲、益智仁、郁金、川芎等合用,针对痰瘀所蒙之清窍受阻,以芳香化浊,祛痰开窍醒神,从而改善痴呆患者头重昏蒙及记忆力下降的作用。组方强调整体观念及辩证论治,遣方配伍精巧,具有整体调治,多环节、多系统、多靶点的优势。
具体地,痴呆之肝肾不足证,可予枸杞子加量至15~20份、益智仁加量至15~20份;痴呆之气虚血瘀证,可予川芎加量至10~20份;痴呆之痰蒙神窍证,可予郁金、石菖蒲加量至15份;痴呆兼见大便秘结者,可予制大黄加量至10份。
优选,按重量计,包含益智仁10份、水蛭3份、石菖蒲12份、枸杞10份、炙黄芪10份、仙鹤草15份、郁金10份、川芎6份和制大黄6份。
作为本发明涉及的第二方面,上述中药组合物的提取物以及药学上可接受的载体形成药物组合物,具体的制剂形式包含颗粒剂、浸膏剂或合剂;当制剂形式为颗粒剂时,上述提取物与药学上可接受的载体的质量比为45~55:55~45。
作为本发明涉及的第三方面,上述药物组合物的制备方法包含以下步骤:
(1)按重量取各中药饮片提取两次;
(2)合并步骤(1)所得提取液,浓缩至60℃相对密度为1.08~1.12的浓缩液;
(3)当药物组合物为颗粒剂时,将步骤(2)所得浓缩液与糊精制粒。
其中,步骤(1)中两次提取的方法分别为第一次提取以中药饮片8~10倍质量的水,浸泡30~60min,煎煮40~60min,滤过;第二次提取以中药饮片6~10倍质量的水,煎煮30~60min,滤过。
步骤(3)中制粒方法为进风温度:80~100℃,喷雾结束后60~70℃继续干燥。
作为本发明涉及的第四方面,上述中药组合物及其药物组合物可制备为预防或/和治疗血管性认知障碍、混合型痴呆或伴有缺血症状的阿尔茨海默症的药物,尤其是针对痴呆引发的肝肾不足、气虚血瘀、痰蒙神窍或大便秘结等证的药物。
有益效果:与现有技术相比,本发明具有如下显著优点:
1、该中药组合物围绕痴呆“毒损脑络”的病理机制,以大黄及水蛭为君药,活血祛瘀、泄毒通络;石菖蒲、益智仁、郁金、川芎等合用,针对痰瘀所蒙之清窍受阻,以芳香化浊,祛痰开窍醒神,从而改善痴呆患者头重昏蒙及记忆力下降;全方以补肾益精、活血通瘀、化痰泄毒为治疗方法,达到改善血管性认知障碍,缓解血管性认知障碍病理学产物聚集的作用,通过各药材的合理配比,协同作用,对治疗血管性认知障碍有明确的治疗作用;
2、该中药组合物具有抗炎功效,通过作用于肠道菌群,抑制有害菌群的分布,增加有益菌群的丰度,降低肠道通透性、减轻肠道炎症;对缺血后脑神经元的损伤具有保护作用,从而改善认知障碍(痴呆型和非痴呆型)患者的临床症状;
3、药材基原稳定易得,无毒副作用;
4、制备方法简便易行,无特殊要求。
附图说明
图1为DP组和CM组治疗前后VaDAS-cog评分比较;
图2为DP组和CM组治疗前后MoCA评分比较;
图3为DP组和CM组治疗前后SDCVD评分比较;
图4为样本稀释曲线;
图5为DP组和CM组治疗前Alpha多样性的相关指数对比;
图6为DP组和CM组治疗前PoCA分析;
图7为中药组治疗前后主要菌门丰度比较;
图8为中药组治疗前后主要菌属丰度比较;
图9为中药组治疗前后LEfSe分析;
图10为多奈哌齐组治疗前后主要菌门丰度对比;
图11为多奈哌齐组治疗前后主要菌属丰度对比;
图12为多奈哌齐组治疗前后LEfSe分析;
图13为CM组、DP组和健康人群PCoA分析;
图14为CM组、DP组治疗后与健康人群菌群模块化的关联网络图;
图15为各组大鼠逃避潜伏时间结果;
图16为各组大鼠定位航行典型游泳轨迹图;
图17为光镜下各组大鼠海马CA1区病理形态观察结果。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1:通脑益智颗粒的制备
取益智仁10份、水蛭3份、石菖蒲12份、枸杞10份、炙黄芪10份、仙鹤草15份、郁金10份、川芎6份、制大黄6份,以上饮片合并,加水煎煮两次。第一次加入饮片重量的10倍量水,浸泡30min,煎煮60min,滤过;第二次加入饮片重量的8倍量水,煎煮60min,滤过,合并滤液,55~65℃下减压浓缩至60℃时相对密度为1.08~1.12,直管式高速离心机离心(转速:16000r/min,流量:4~6L/min)。取糊精适量,置流化床中,设置进风温度为100℃,当物料温度升至70℃时,开始进液,进液速度控制在80~150r/min,雾化压力外0.2MPa,内0.15MPa。喷雾结束,60~70℃继续干燥1~1.5h,共制成1000g。
实施例2:通脑益智颗粒的临床疗效及对患者肠道菌群的影响
1、实验资料
1.1研究样本
选择在2020年04月至2020年12月期间就诊于南京中医药大学附属医院门诊及病房的患者,既往有脑血管病史,经由专业受培训过的人员进行MoCA、CDR、HIS量表评估后,筛选出符合纳入标准的病人,再进行血管性痴呆评估量表(Vascular dementia assessmentscale-cog,VaDAS-cog)及血管性认知障碍的中医辨证量表(SDSVD)的评估。
参考《临床试验样本含量的计算》以及统计学专业人员进行小样本随机对照试验的估算,结合前期试验结果,确定样本量为40例。通过SAS软件产生随机号,将40例患者随机分配到中药组和对照组。主要告知受试者及其直系家属本研究的目的、主要内容以及参与本研究潜在的获益和风险情况,并由本人或其家属签署知情同意书,配合随访要求。本研究已通过南京中医药大学附属医院伦理委员会审核(2020NL-104-02)。
1.2诊断标准
1.2.1西医诊断标准
(1)按照类肝素药物治疗急性缺血性脑卒中试验(TOAST)亚型分类标准选择小动脉闭塞性卒中或腔隙性卒中(SAA)患者,临床及影像学表现符合以下3项标准之一:①有典型的腔隙性梗死的临床表现,影像学检查有与临床症状相对应的责任病灶且最大直径<1.5cm;②具有非典型的腔隙性梗死的表现,而影像学检查后发现与临床症状相符的<1.5cm的责任病灶;③临床上有典型的腔隙性梗死的症状,但影像学上未发现有相对应的责任病灶。
(2)非痴呆型血管性认知功能障碍诊断标准参照《2007年血管性认知功能损害的专家共识》指定:①不符合痴呆标准,临床痴呆评定量表(clinical dementia rating,CDR)的评分为0.5分,蒙特利尔认知评估量表(Montreal Cognitive Assessment,MoCA)得分<26分,Hachinski(HIS)缺血量表≥7分;②认知损害被认为是血管性,有突然起病、阶梯样病程、斑片状认知损害的证据;③有动脉粥样硬化证据、局灶体征和影像学证据;④有血管性危险因素,但不含仅有血管性危险因素而无梗死/缺血体征者。
1.2.2中医诊断标准
参考2002年版《中药新药临床研究指导原则》及田金洲等研究人员订制的血管性认知障碍的中医辨证量表(SDSVD)制定肾精亏虚、痰瘀阻络证诊断标准。①主症:主症:智能减退(思维呆滞、善忘无记、情感改变、语言错乱);②次症:腰膝酸软、善惊易恐、头晕头痛、耳鸣脑鸣、纳呆脘胀、神疲乏力、肢体困重、面部发麻、失眠烦躁;③舌脉:舌质暗红或紫,或有瘀点、瘀斑,苔薄白或腻,脉沉、弦、细、涩或无力。符合上述主症且具有次症中的任意2项及以上者,结合舌脉,方可进行明确诊断。
1.3纳入标准
(1)年龄在50~80岁;
(2)符合中西医诊断标准;
(3)受试者:①健康组:无脑血管疾病史、无认知功能损害、无重要脏器损害、平素体检正常者。②非痴呆型血管性认知障碍组:符合血管性认知功能障碍诊断标准的患者,CDR为0.5分,14<MoCA得分<26分;
(4)患者或患者监护者对研究意义有正确认识,对研究人员的观察和评价有良好的依从性;
(5)有一定文化程度(既往能阅读简单的报纸文章,可书写小文章);
(6)患者或患者监护者知情同意,志愿受试,并签署相关同意书。获得知情同意书过程应符合GCP规定。
1.4排除标准
(1)血管炎、静脉及静脉窦血栓形成;
(2)抑郁症或其他精神障碍;
(3)患有某些能干扰认知功能评价的疾病,其中包括嗜酒、头部外伤、吸毒或其他精神性药物滥用者;
(4)伴有严重的神经功能缺损者,如严重偏瘫、各种失语、视听障碍等;
(5)慢性具有严重的呼吸系统疾病、原发性心血管病变、肝脏病变、肾脏病变、血液系统病变、糖尿病、消化系统疾病,或影响其生存的严重疾病,如肿瘤或艾滋病等;
(6)根据研究者的判断、具有降低入组可能性或使入组复杂化的其他病变,如工作环境经常变动等易造成失访的情况;
(7)1个月内服用过任何抗生素或益生菌制剂及质子泵抑制剂者;
(8)处于急性疾病状态(如发热、肺炎、急性脑梗塞等);
(9)免疫状态不佳及肠道粘膜严重受损者或患有其他可能影响肠道动力及菌群疾病者;
(10)目前正在服用改善认知障碍的药物(包括中药和西药)。
1.5主要实验试剂与仪器
(1)实验主要试剂
(2)实验主要仪器
2、研究方法
2.1实验分组
将VCIND人群按照随机号随机分为中药治疗组(Chinese medicine,CM)和对照组(多奈哌齐治疗组Donepezil,DP)。CM治疗组治疗前后分别为命名为CM-1和CM-2;DP治疗组治疗前后分别命名为DP-1和DP-2。
2.2治疗方法
2.2.1基础治疗
(1)控制血压:钙离子通道阻断剂、血管紧张素转换酶抑制剂等能控制血压且对大脑认知功能没有损伤的降压药;
(2)控制血脂:他汀类药物如瑞舒伐他汀、阿托伐他汀等可以降低胆固醇,稳定动脉硬化斑块的药物;
(3)脑梗塞患者可采用抗血小板聚集药物治疗,如:阿司匹林、氯吡格雷等抑制血小板的聚集,防止血栓形成,改善脑循环;
(4)冠心病患者可服用改善心肌缺血的药物:如单硝酸异山梨酯、曲美他嗪等可改善心肌供血药物;
(5)健康教育:予清淡、易吞咽、易消化、低胆固醇饮食,少量多餐,多听广播、音乐,多看书、读报,家属加强安全护理意识,防止摔伤、外出不归等意外发生。
2.2.2试验用药
中药组:通脑益智颗粒(制大黄6份,郁金10份,炙黄芪10份,川芎6份,石菖蒲12份,枸杞10份,水蛭3份,益智仁10份,仙鹤草15份);
对照组:根据公认有效、安全、可比原则,选择多奈哌齐(安理申,卫材药业),规格:H20050978。
2.2.3用药方法
对照组:给予基础治疗+多奈哌齐5mg,1次/晚,共使用90天;
中药组:给予基础治疗+通脑益智颗粒治疗,早晚各1剂,共使用90天。
2.2.4合并用药
除试验用药外,观察期间禁止使用其它治疗认知障碍的中药和西药,以及与本病治疗相关的,影响肠道微生态的治疗如益生菌等。
2.3临床观察项目
2.3.1生物学指标
(1)人口学特征:性别、年龄、BMI、基础病史等;
(2)生命体征:体温,静息心率,呼吸,休息10分钟后的血压。
2.3.2疗效观察指标
(1)主要指标:治疗前后肠道菌群及SCFAs变化(注:肠道菌群检测粪便收集:收集清晨成形粪便(样本量约5g),置于50mL无菌粪便采集器中。粪便标本放置在无菌低温环境下,并于2小时内送往实验室-80度冰箱内储存);
(2)次要指标:认知功能:VaDAS-cog、MMSE量表,中医证候评分量表:SDCVD(参照《中药新药临床研究指导原则》及田金洲血管性认知障碍的中医辨证量表制定具体见附录)。
2.3.3安全性观察指标
心电图、血生化、血常规、尿常规、粪常规。
2.3.4观察节点
(1)主要症状体征:初诊首日,第1月、2月、3月各观察随访记录1次。治疗前后均留取粪便做菌群检查;
(2)实验室各项疗效性、安全性检查项目治疗前后各做1次。
2.4疗效评价
(1)疗效指数=(治疗后得分—治疗前得分)/治疗前得分×100%(适用于得分增加为有效的指标),显效:≥20%,有效:≥12%,无效:<12%,恶化:<-12%;
(2)疗效指数=(治疗前得分—治疗后得分)/治疗前得分×100%(适用于得分减少为有效的指标),显效:≥20%,有效:≥12%,无效:<12%,恶化:<-12%。
2.5肠道菌群测序分析
2.5.1样本DNA抽提
2.5.2引物设计与合成
选择V3-V4区作为16S rDNA扩增,通用引物为338F和806R。在引物的5’端加上适合测序的index序列和接头序列,完成特异性引物的设计与合成:
Forward premier:5’-ACTCCTACGGGAGGCAGCAG-3’
Reverse premier:5’-GGACTACHVGGGTWTCTAAT-3’
2.5.3 PCR扩增和产物纯化
使用已合成引物进行PCR扩增,扩增程序为:95℃预变性3min,27个周期(95℃变性30s,55℃退火30s,72℃延伸30s),最后72℃延伸10分钟。扩增系统为20μL,4μL 5*FastPfu缓冲液,2μL 2.5mM dNTPs,0.8μL引物(5μM),0.4μL FastPfu聚合酶;10ng DNA模板。
向PCR产物中加入0.8倍体积的磁珠,摇匀并完全悬浮,在磁性框架上吸附5分钟,小心地吸出上清;加入20μL 0.8倍磁珠洗涤液,震荡充分悬浮后放在磁力架上吸附5min,小心吸出上清;加入20μL 0.8倍的磁珠清洗液,摇匀充分悬浮,吸附在磁框上5分钟,小心吸出。加入200μL 80%乙醇,反放在磁框上,用磁珠吸附在PCR管的另一侧,充分吸附后吸出上清液。然后在室温下放置5分钟,直到酒精完全挥发,磁珠破裂。加入25μL Elution Buffer洗脱;将PCR管放在吸附架上5分钟,完全吸附,从上清取出,存放在干净的1.5mL离心管。
2.5.4 PCR产物定量
PCR扩增回收的产物进行荧光定量。荧光试剂为Quant-iTPicoGreen dsDNA AssayKit,定量仪器为Microplatereader(BioTek,FLx800)。根据荧光定量结果,按照每个样本的测序量需求,对各样本按相应比例进行混合。
(1)通过试剂盒中的末端修复混合物去除DNA序列5'末端的突出碱基,并添加磷酸盐基团以补充3'末端缺失的碱基;
(2)A碱基被添加到DNA序列的3'端,以防止DNA片段自我连接,并确保目标序列可以连接到测序连接器;
(3)在序列的5'末端添加含有库特异性标签(即Index序列)的测序连接器,以使DNA分子固定在流动细胞上;
(4)采用BECKMAN AMPure XP Beads,通过磁珠筛选去除接头自连片段,纯化添加接头后的文库体系;
(5)对上述连上接头的DNA片段进行PCR扩增,从而富集测序文库模板,并采用BECKMAN AMPure XP Beads再次纯化文库富集产物;
(6)通过2%的琼脂糖凝胶电泳对文库的最终片段进行筛选和纯化。
2.5.5上机高通量测序
(1)测序前,需要对文库进行质检,采用Agilent High Sensitivity DNAKit。
合格的库有且只有一个没有接头的单峰。
(2)采用Quant-iTPicoGreen dsDNA Assay Kit在PromegaQuantiFluor荧光定量系统上对文库进行定量。
(3)将合格的各上机测序文库(Index序列不可重复)梯度稀释后,根据所需测序量按相应比例混合,并经NaOH变性为单链进行上机测序。
2.5.6数据分析流程
为确保研究质量,在进行数据分析前要将所有原始数据进行过滤处理。首先调用qiimecutadapttrim-paired切除序列的引物片段,弃去未匹配引物的序列;然后通过qiimedada2denoise-paired调用DADA2进行质控、去噪、拼接、去嵌合体。DADA2方法主要进行去引物,质量过滤,去噪(denoise),拼接和去嵌合体等步骤。不再以相似度聚类,只进行去重(dereplication)或者说相当于以100%相似度聚类。使用DADA2质控后产生的每个去重的序列称为ASVs(amplicon sequence variants),或称为特征序列(对应于OTU代表序列),而这些序列在样本中的丰度表称为特征表(对应于OTU表)。将过滤后的优化序列进行操作分类单元的聚类,根据聚类的结果进行细菌分类学分析。基于分类学信息,在各个分类水平上进行群落结构的统计分析。
2.5.7质谱法测定肠道菌群代谢物SCFAs
(1)标曲配置:测适量乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸和己酸纯标准品适量,用乙醚配制成0.02μg/mL,0.1μg/mL,0.5μg/mL,2μg/mL,10μg/mL,25μg/mL,50μg/mL,100μg/mL,250μg/mL,500μg/mL,10个混合标准浓度梯度。母液及工作标准溶液均保存于-20℃。
(2)代谢物提取:将混标浓度为25μg/mL的标准样品连续进样八次,计算日内精密度,以RSD表示。每天处理一份25μg/mL标准样品于第一天、第二天、第三天测定,计算日间精密度,以RSD表示。日内精密度在1.00%~1.85%之间,日间精密度在4.76%~11.73%之间,表明仪器的精密度良好。
取适量样本于2mL离心管中,加50μL 15%磷酸,再加125μg/mL的内标(异己酸)溶液100μL和乙醚400μL匀浆1min,于4℃12000rpm离心10min,取上清上机测试。
(3)上机检测:色谱条件:色谱柱Agilent HP-INNOWAX毛细管柱(30m*0.25mm ID*0.25μm);分流进样,进样量1μL,分流比10:1。进样口温度250℃;离子源温度300℃;传输线温度250℃。程序升温起始温度90℃;然后以10℃/min升温至120℃;再以5℃/min升温至150℃;最后以25℃/min升温至250℃维持2min。载气为氦气,载气流速1.0mL/min。
(4)质谱条件:电子轰击电离(EI)源,SIM扫描方式,电子能量70eV。
2.6统计学处理
统计分析将采用IBM SPSS21.0统计分析软件进行计算。所有的统计检验均采用双侧检测,P值小于或等于0.05将被认为所检验的判别有统计意义。不同治疗组各次就诊的计量资料将采用进行统计描述。与筛选期基础值进行比较,采用配对t检验比较组内前后差异。两组治疗前后的变化采用方差分析(ANOVA)进行比较。不同治疗组各次就诊的计数资料采用频数进行统计描述。两组治疗前后的变化采用卡方检验或非参数检验。
3、研究结果
3.1研究完成情况
所有受试者均为符合纳入标准、排除标准的非痴呆型血管性认知障碍患者,按照研究方案,本研究受试者共纳入40例,其中中药组共纳入20例,对照组共纳入20例,本次纳入的病例依从性良好,无脱落病例,均完成了全程受试流程。
3.2受试人群基线情况
对随机分入两组的临床一般基线资料进行统计学分析,根据数据的正态性、方差齐性选择不同的统计学方法。人口学资料上,两组在年龄、性别、心率、BMI、受教育年限、每周规律运动时间<2h上无明显差异(P>0.05);在个人生活史上,两组间在吸烟史、饮酒史上对比无明显差异(P>0.05);在个人病史方面,两组在高血压病史、糖尿病病史、心血管疾病史方面具无显著差异(P>0.05);而在关于病情和认知的评分量表上两组SDCVD评分、MMSE评分和VaDAS-cog评分在治疗前差异均无统计学意义(P>0.05)。两组患者基线资料具有可比性,具体信息见表1。
表1.两组人群基线资料对比
3.3临床疗效评价
3.3.1治疗前后各认知量表对比
(1)治疗前后两组VaDAS-cog评分量表比较:治疗前,两组患者VaDAS-cog评分组间比较无显著差异(P=0.690),具有可比性;治疗后两组患者VaDAS-cog评分较治疗前均下降,但治疗后两组组间对比无明显差异(P=0.379)。结果详见表2,图1。
注:*为两组组间比较,+为两组各自治疗前后组内比较。
(2)治疗前后两组MMSE评分量表比较:治疗前,两组患者MMSE评分组间比较无显著差异(P=0.088),具有可比性;治疗后两组患者MMSE评分较治疗前均上升,但治疗后两组组间对比无明显差异(P=0.490)。结果详见表3,图2。
注:*为两组组间比较,+为两组各自治疗前后组内比较。
3.3.2治疗前后两组中医证候症状积分对比
治疗前后两组中医证候症状积分比较:治疗前,两组患者SDCVD量表评分组间比较无显著差异(P=0.818),具有可比性;治疗后两组患者SDCVD评分较治疗前均下降,但治疗后两组组间对比无明显差异(P=0.266)。结果详见表4,图3。
注:*为两组组间比较,+为两组各自治疗前后组内比较。
3.3.3两组综合疗效评价
根据结果计算显示:CM组治疗总有效率是75%,DP组治疗总有效率为80%,利用卡方检验对两组进行分析,CM组和DP组较治疗前有好转,两种治疗方法对患者病情均有改善,但两组之间差异没有统计学意义(P=0.50),提示通脑益智颗粒的疗效与多奈哌齐相当。结果详见表5。
表5.两组综合疗效评价
3.4肠道菌群测序结果分析
3.4.1物种稀释曲线
首先需要判断本次样本测序数据量是否足够,从样本中随机抽取一定测序量数据,统计物种数目,然后用测序数量和物种多样性来构建稀释曲线,如下图4,本次测序各样本的稀释曲线趋于平缓,说明本研究中各样本抽取的测序数据量已足够合理,如果继续加大数据量,可能也不会产生更多的物种数。
3.4.2治疗前中药组和多奈哌齐组肠道菌群差异
(1)分析治疗前两组Alpha多样性的相关指数,包括Observed species指数、Chao1指数、Shannon指数、Simpson指数等。结果显示:两组在治疗前肠道菌群的Observedspecies指数、Chao1指数对比无明显差异(P=0.36,0.61>0.05),提示两组的物种丰度相似;两组肠道菌群Shannon指数、Simpson指数对比无显著性差异(P=0.36,0.79>0.05),提示两组物种的多样性和均匀度相似。结果详见图5。
(2)对治疗前的两组进行Beta多样性分析,比较两组样本间的微生物群落构成。先使用了unweighted unifrace距离进行PCoA分析,并选取贡献率最大的主坐标组合进行作图展示,图中样品的距离越近,表明样品的物种组成结构越相似。之后结合Anosim分析共同判断这两组样本Beta多样性是否有差异。
具体分析图见下图6所示,以Axis1(9%)为横坐标,Axis2(6.4%)为纵坐标,建立PCoA图,观察中药组和多奈哌齐组在治疗前两组内各个样本在图上两两之间的距离差异不显著,结合Anosim分析结果P=0.199,提示在治疗前中药组(CM)和多奈哌齐组(DP)组间物种多样性差异不明显。
通过分析治疗前中药组与多奈哌齐组肠道菌群多样性、物种差异,得出结果认为两组的菌群在结构和丰度上差异较小,具有可比性。
3.4.3中药组治疗前后肠道菌群差异
(1)中药组治疗前后肠道菌群Alpha多样性分析
比较治疗之前后中药组Alpha多样性的差异。结果显示:经治疗后中药组的Observed species指数、Chao1指数较治疗前无明显统计学差异(P=0.303,0.285),表明治疗后物种丰富度无明显变化,虽然在统计学上无差异,但发现治疗后中药组的丰富度有上升趋势;此外治疗后Shannon指数较前增长且差异具有统计学意义(P=0.002),Simpson指数差异不显著(P=0.759),提示中药治疗后肠道菌群多样性和均匀度增加。结果详见表6。
注:CM-1指治疗前,CM-2指治疗后。
(2)中药组治疗前后肠道菌群物种变化
为进一步了解治疗前后,中药组肠道菌群物种构成,探寻在不同分类水平上物种的相对丰度差异,进行了两组间不同分类水平上的物种差异分析。
①中药组治疗前后肠道菌群在门水平上相对丰度变化结果:厚壁菌门(79.60%vs75.69%)、拟杆菌门(9.74%vs 12.51%)、放线菌门(7.55%vs 3.48%)、变形菌门(4.68%vs 2.98%)、疣微菌门(1.49%vs 0.45%)、梭杆菌门(0.41%vs 2.34%)、蓝藻菌门(0.14%vs 0.01%)。中药组治疗前后相比厚壁菌门、变形菌门、放线菌门、疣微菌门、梭杆菌门等菌门相对丰度均无显著性差异,而治疗后较治疗前拟杆菌门丰度上升,蓝藻菌门下降,差异有统计学意义。结果详见表7,图7。
表7.中药组治疗前后主要菌门丰度比较分析
②中药组治疗前后肠道菌群在属水平上相对丰度变化结果:治疗前中药组按照相对丰度依次排序前十位是普拉梭菌属(11.5%)、拟杆菌属(8.41%)、布劳特氏菌属(7.26%)、罗氏菌属(6.71%)、疣微菌属(5.72%)、毛螺菌属(4.06%)、粪球菌属(4.13%)、梭菌属(3.14%)、双歧杆菌属(2.91%)、链球菌属(2.79%);治疗后中药组相对丰度前十位的菌属是普拉梭菌属(17.03%)、拟杆菌属(9.60%)、布劳特氏菌属(8.73%)、双歧杆菌属(6.92%)、疣微菌属(4.72%)、毛螺菌属(3.99%)、瘤胃球菌属(3.76%)、芽殖菌属(3.54%)、乳酸杆菌属(3.17%)、粪球菌属(3.03%)。在相对丰度较高的菌属种,中药组经治疗后普拉梭菌属、双歧杆菌属、乳酸杆菌属、瘤胃球菌属丰度明显升高,差异较治疗前有统计学差异(P=0.006,0.023,0.001,0.048)。结果详见图8。
(3)中药组治疗前后物种差异分析
为了进一步探寻中药组治疗前后组间肠道菌群物种差异在各分类水平上的物种单元,应用LEfSe分析,以LDA>3.0为临界值。结果显示如下,中药组治疗前后肠道菌群发生变化,其中有特异性的分类单元包括:治疗前有Lactobacillaceae、Lactobacillus等11个有特异性的物种单元明显增多;治疗后有Rhodobacter、Phascolarctobacterium等4个单元明显增多。结果详见图9。
3.4.4多奈哌齐组治疗前后肠道菌群差异
(1)多奈哌齐组治疗前后肠道菌群Alpha多样性分析
比较多奈哌齐组治疗之前后Alpha多样性的差异。结果显示:经治疗后DP组的Observed species指数、Chao1指数、Shannon指数和Simpson指数较治疗前均无明显变化,且在统计学上均不存在差异(P>0.050.303),表明经DP治疗后改组肠道菌群的物种丰富度、多样性和均匀度均无明显变化。具体见表8。
注:DP-1指治疗前,DP-2指治疗后。
(2)多奈哌齐组治疗前后肠道菌群物种变化
①多奈哌齐组组治疗前后肠道菌群在门水平上相对丰度变化结果:厚壁菌门(76.71%vs 81.09%)、拟杆菌门(8.64%vs 9.61%)、放线菌门(7.57%vs 5.53%)、变形菌门(4.66%vs 2.43%)、疣微菌门(0.47%vs 1.05%)、梭杆菌门(1.49%vs 0.24%)、蓝藻菌门(0.49%vs 0.27%)。多奈哌齐组治疗前后相比,厚壁菌门、拟杆菌门、变形菌门、放线菌门、疣微菌门等菌门相对丰度均无显著性差异;而治疗后较治疗前梭杆菌门和蓝藻菌门相对丰度下降,差异有统计学意义。结果详见表9,图10。
表9.中药组治疗前后主要菌门丰度比较分析
②多奈哌齐组治疗前后肠道菌群在属水平上相对丰度变化结果:治疗前DP组按照相对丰度依次排序前十位是普拉梭菌属(9.0%)、链球菌属(7.36%)、乳杆菌属(7.19%)、布劳特氏菌属(6.25%)、罗氏菌属(6.25%)、拟杆菌属(6.05%)、疣微菌属(5.53%)、双歧杆菌属(4.40%)、毛螺菌属(3.47%)、粪球菌属(3.13%);治疗后中药组相对丰度前十位的菌属是普拉梭菌属(15.02%)、罗氏菌属(10.20%)、疣微菌属(5.76%)、布劳特氏菌属(5.49%)、拟杆菌属(5.17%)、链球菌属(5.03%)、双歧杆菌属(4.77%)、粪球菌属(4.63%)、毛螺菌属(3.30%)、瘤胃球菌属(2.95%)。在相对丰度较高的菌属种,DP组经治疗后普拉梭菌属丰度明显升高(P=0.015),乳酸杆菌属丰度明显下降(P=0.002),差异较治疗前有统计学差异。结果详见图11。
(3)多奈哌齐组治疗前后物种差异分析
应用LEfSe分析,以LDA>3.0为临界值。探索DP组治疗前后组间肠道菌群物种差异在各分类水平上的物种单元。结果显示如下,DP组治疗前后肠道菌群发生明显变化,其中有特异性的分类单元包括:治疗前有Bacilli、Lactobacillales、Streptococcaceae等30个有特异性的物种单元明显增多;治疗后有Veillonellaceae、Bifidobacteriaceae、Bifidobacteriaceae等13个单元增多。结果详见图12。
3.4.5中药组和多奈哌齐组治疗后与健康人群肠道菌群的关联
为进一步探寻经中药及多奈哌齐治疗后两组患者肠道菌群的变化趋势,以及不同样本之间微生物群落空间构成进行比较,以及不同治疗手段导致肠道不同环境的过程,是否导致了该群落物种装配(assembly)的差异。尝试进行了以下两种分析。
(1)Beta多样性分析
具体分析图见下图13所示,以Axis1(34.4%)为横坐标,Axis2(11.3%)为纵坐标,建立PCoA图,观察中药组和多奈哌齐组在治疗后与健康人群,在图上两两之间的距离差异,图中给出了三组PCoA物种多样性的分析结果,DP组和健康人去组除少数样本有交叉外,组间差异较明显,而CM组与健康人群样本相似。DP组样本主要分布在第四象限,而CM组和健康人群的样本主要分布在第一、二、三象限。HC组主要分布在上半象限。结合Anosim两两分析结果(P=0.236,0.048),提示在治疗后中药组(CM)人群的肠道菌群向健康人群靠近,而多奈哌齐组(DP)组肠道菌群物种没有这种趋势。
(2)关联网络分析
利用R软件基于微生物成员之间相互关系的网络推断分析(network analysis)来探寻微生物群落之间的联系。通过关联分析的方法,寻找探析微生物群落在时空变化,药物代谢过程驱动下所呈现的共现(Co-occurrence)或互斥(Co-exclusion)的现象,从而明确微生物类群是否存在特定的生态功能或者外来药物的干预能否撬动整个群落的组成变化。一般来说,在微生物生态学的关联网络图中,每个点可称为一个node(或vertex),它可以代表群落中的一个ASV/OUT,又或是一个分类单元;两个点之间的连接线可称为edge,代表所连接的两个点之间的分布趋势。
通过分析发现,中药治疗组经治疗后菌群与健康人群菌群关联密切,表明通脑益智颗粒可以改变VCIND患者的生态群落;而在多奈哌齐组中菌群的结构与中药组以及健康人群的没有见到明显的关联。结果详见图14。
3.5肠道菌群代谢物-短链脂肪酸定量分析
通过质谱定量分析,对检测到的所有色谱峰使用标准质谱库进行识别,再通过与对照品进行对照,结果共在两组患者鉴定出7种SCFAs,分别为乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸和己酸。其中乙酸、丙酸和丁酸是肠道菌群代谢物-短链脂肪酸的优势物种,占比最高。两组患者在治疗前的肠道内短链脂肪酸的基线数据相近,二者具有可比性。
(1)中药组SCFAs治疗前后变化定量分析
中药组患者治疗后较治疗前,乙酸、丙酸、丁酸明显上升,差异具有统计学意义(P=0.006,0.023,0.029);而异丁酸、异戊酸、戊酸和己酸较治疗前无明显改变。提示通脑益智颗粒改善VCIND患者认知功能的作用,可能与其调节肠道菌群代谢物-短链脂肪酸的增加有关。结果详见表10。
注:CM-1指中药组治疗前,CM-2指治疗后。
(2)多奈哌齐组SCFAs治疗前后变化定量分析
多奈哌齐组患者的肠道菌群代谢物SCFAs治疗后较治疗前,乙酸、丁酸有上升,但差异无统计学意义(P=0.467,0.347);异丁酸、异戊酸、戊酸和己酸较治疗前无明显改变。经治疗后DP组与CM组的SCFAs相比无统计学意义。提示多奈哌齐药物对VCIND患者肠道菌群改变不明显。结果详见表11。
注:DP-1指多奈哌齐组治疗前,DP-2指治疗后。
3.6安全性分析
两组患者在治疗前后的的心电图、血常规、尿常规、粪常规、肝功能、肾功能均无明显改变,组间比较无差异,无统计学意义。但在用药期间,中药组有2例患者出现腹泻症状,未经干预后自然好转;而多奈哌齐组有1例患者出现夜晚恶梦增多现象,对其进行心理疏导后有好转。另外1例多奈哌齐组患者出现失眠现象,未对其使用药物干预,对其进行睡眠卫生习惯教育后有好转。余未见明显不良事件发生。
4、结论
从肠道菌群多靶点的角度来探寻通脑益智颗粒的临床疗效。通过对非痴呆型血管性认知障碍患者肠道菌群的检测,以及服用通脑益智颗粒和多奈哌齐的VCIND患者进行肠道菌群以及代谢物的检测,明确VCIND患者的肠道菌特征,证明肠道菌群及其代谢物的改变影响了VCIND的发病过程。
中药组和多奈哌齐组治疗后的认知量表和中医证候积分较治疗前均有积极改善,两组之间比较无显著差异。经多奈哌齐治疗后的VCIND患者肠道菌群丰度和多样性无明显改变,但有益菌乳酸杆菌属的丰度明显下降。而经通脑益智颗粒治疗后,VCIND患者的普拉梭菌属、双歧杆菌属、乳酸杆菌属、瘤胃球菌属丰度以及短链脂肪酸有明显的提高,而这可能会让肠道通透性降低、肠道炎症减轻及神经保护增加,最终改善了VCIND患者的临床症状。
总之,在改善临床认知症状方面,通脑益智颗粒与临床经典药物多奈哌齐相比,两者疗效相当;通脑益智颗粒还可以使肠道有益菌的丰度和数量增加,达到神经保护作用,起到中医学“标本兼治”的功效。
实施例3:Morris水迷宫实验
1、动物模型制作
将健康3月龄雄性SD大鼠预先进行Morris水迷宫检测,根据大鼠的游泳速度及相关游泳姿态怪异程度,剔除不合格大鼠。随机分组,为后期分别造模做准备。
采用改良式双侧颈动脉结扎的方式制作模型大鼠:术前大鼠禁食12h,不禁饮水。假手术组用3%的戊巴比妥钠(1.5mL/kg)腹腔内注射方式以麻醉大鼠,把大鼠于固定在操作台上,以仰卧方式固定绑牢,避免损伤大鼠四肢,用小剪刀剃除或刮毛刀刮除颈部皮毛,面积约为一元硬币大小,手术途中避免破损皮肤,常规碘伏消毒后手术刀切开颈部,暴露颈部肌肉,钝性剥离结缔组织和颈前肌群,不予以切断血管操作,缝合大鼠颈部伤口,敷以青霉素药粉。对模型组大鼠同样予以腹腔内注射3%的戊巴比妥钠(1.5mL/kg)方式以麻醉大鼠,仰卧位固定于操作手术台之上,用小剪刀剃除或刮毛刀刮除颈部皮毛,面积约为一元硬币大小,手术途中避免破损皮肤,常规碘伏消毒,取大鼠颈项正中部位,采用手术刀切口,充分暴露颈部肌肉,保持切口整齐,予以扁平状镊子钝性剥离大鼠的结缔组织和其颈前肌群,以达到暴露颈部血管,用玻璃棒精细分离与血管粘连的迷走神经,避免损伤迷走神经,同时达到分离大鼠两侧颈总动脉目标,以“0”号手术用丝线分别结扎大鼠颈总动脉近端和远端,并用剪刀从结扎中间部位剪断血管,以达到永久阻断颈总动脉血管血流供脑目的。伤口处同样适量洒青霉素粉,后分步缝合内外层伤口。在手术过程中,始终观察动物呼吸运动频率,保持动物气道通畅,能够自主呼吸,并始终保持大鼠肛温在37±0.5℃。
2、大鼠分组及给药
造模1周后,查看大鼠手术部位切口,愈合完全达到预定目标,开始随机分组。术后1周内共死亡大鼠4只,假手术组死亡大鼠1只,剩余11只假手术类大鼠;模型组死亡3只大鼠,剩余存活大鼠55只;后进行相关水迷宫行为学测试,剔除不符合标准大鼠4只,筛选出造模成功的大鼠:50只血管性痴呆模型大鼠和10只假手术大鼠,其中假手术组单独组成假手术组(A),将剩下血管性痴呆大鼠随机分为5组,每组10只,分别为模型组(B)、多奈哌齐组(C)、通脑益智颗粒低剂量组(D)、通脑益智颗粒中剂量组(E)、通脑益智颗粒高剂量组(F)。
假手术组及模型组:每天给予等量生理盐水作为对照。
多奈哌齐组:予多奈哌齐1mg/kg/天灌胃给药,连续给药4周;
通脑益智颗粒低剂量组:予通脑益智颗粒4.5g/kg/天灌胃给药,连续给药4周;
通脑益智颗粒中剂量组:予通脑益智颗粒9g/kg/天灌胃给药,连续给药4周;
通脑益智颗粒高剂量组:予通脑益智颗粒18g/kg/天灌胃给药,连续给药4周。
依据人与老鼠身体表面积系数比换算来配置给药分量,中剂量组以临床人用药等效剂量换算为9g/kg,低剂量组为4.5g/kg,高剂量组为18g/kg给药。
采用Morris水迷宫实验进行学习记忆能力检测。
3、Morris水迷宫实验
人为将水迷宫的水池平均划分为四个相等的分象限,分别标记,并且需要在水池平台内壁处标记4个象限入水点;另外在水迷宫平台内壁贴有四个不同形状的图形;将平台安置于水池内第三象限中心地带。实验前向水池内注水,水位要高过平台2cm左右。将墨汁加入水中,将墨汁充分搅匀,以达到隐藏平台目的。给水池内的水加温,并始终维持在21℃左右。水池顶部安装有一部固定式小型追踪摄像机,以追踪并记录大鼠实验时游泳轨迹,进行定位航行实验和空间探索实验。
4、实验结果
(1)定位航行实验结果
表12.各组大鼠在不同时间段定位航行平均逃避潜伏期比较(Mean±SD)
注:A:假手术组;B:模型组;C:多奈哌齐组;D:通脑益智颗粒低剂量组;E:通脑益智颗粒中剂量组;F:通脑益智颗粒高剂量组与假手术组相比:#P<0.01;与模型组相比:*P<0.01。
如表12、图15所示,在定位航行实验中,随着训练时间的延长,各大鼠的训练潜伏期均成递减趋势。与假手术组作比,从第2天开始痴呆模型组大鼠的逃避潜伏期明显延长(P<0.01),与模型组作比,多奈哌齐组与通脑益智颗粒高、中剂量组逃避潜伏期显著缩短(P<0.01),运动距离也明显增加,说明大鼠相对活跃。通脑益智颗粒低剂量组的逃避潜伏期同样缩短,但不如通脑益智颗粒中、高剂量组明显,与药物剂量呈正相关。如图16所示,从第五天的航行轨迹看,假手术组大鼠游泳线轨迹清晰、能够在较短时间及距离内找到水下隐藏的平台。模型组大鼠在寻找水下隐藏平台时,路线是混乱的、大鼠盲目因而不知方位。最终在规定的时间内大鼠也未能找出平台位置。与模型组对比,多奈哌齐组和通脑益智颗粒高剂量组大鼠找寻平台之前的游泳距离缩短,在平台有效区域停留时间相对变长,找寻目标平台的学习记忆能力得到一定改善。
(2)空间搜索实验结果
表13.各组大鼠空间探索结果(Mean±SD)
注:A:假手术组;B:模型组;C:多奈哌齐组;D:通脑益智颗粒低剂量组;E:通脑益智颗粒中剂量组;F:通脑益智颗粒高剂量组(#P<0.01,##P<0.05,*P<0.01)。
如表13中所示,在大鼠第五天的空间搜索实验中,与假手术组相比,模型组大鼠在运动总距离、平台象限停滞时间、有效区域进如次数明显减少(#P<0.01)。而与模型组痴呆大鼠相比,通脑益智颗粒低剂量组大鼠在运动总距离、平台象限停滞时间,穿越台次数上存在显著性差异(##P<0.05);通脑益智颗粒中、高剂量组在运动总距离、平台象限停滞时间,穿越台次数有显著性差异有差异(*P<0.01);多奈哌齐组与通脑益智颗粒中剂量组相比,在运动总距离,穿越平台次数无统计学意义,在平台象限停滞时间有统计学意义(P<0.05)。
(3)治疗后各组大鼠海马CA1区病理观察结果
采用光学显微镜观察了大鼠海马的CA1区域。如下图17所示,sham组大鼠海马CA1区切片中锥体神经元排列紧密有序,细胞形态正常,细胞内结构完整,核膜明显。VCI组大鼠海马CA1区出现神经元萎缩、神经元丢失、神经元排列松散、神经元形态不规则等神经病理损害。在用通脑益智颗粒多种剂量和多奈哌齐治疗后的大鼠中,观察到神经元的损伤较VCI组有改善,少有细胞坏死现象。
该研究结果表明通脑益智颗粒和多奈哌齐均可以减轻了慢性脑缺血所致大鼠认知障碍的病理损伤,各治疗组神经元丢失现象均有改善,但通脑益智颗粒高剂量组改善的更明显。
Claims (10)
1.一种通脑益智的中药组合物,其特征在于,按重量计,包含益智仁10~20份、水蛭3~10份、石菖蒲10~25份、枸杞10~20份、炙黄芪10~30份、仙鹤草12~30份、郁金5~15份、川芎5~20份和制大黄5~10份。
2.根据权利要求1所述的中药组合物,其特征在于,按重量计,当应用于痴呆之肝肾不足证时,枸杞子用量为15~20份、益智仁用量为15~20份;当应用与痴呆之气虚血瘀证时,川芎用量为10~20份;当应用于痴呆之痰蒙神窍证时,郁金、石菖蒲用量为15份;当应用于痴呆兼见大便秘结证时,制大黄用量为10份。
3.根据权利要求1所述的中药组合物,其特征在于,按重量计,包含益智仁10份、水蛭3份、石菖蒲12份、枸杞10份、炙黄芪10份、仙鹤草15份、郁金10份、川芎6份和制大黄6份。
4.一种药物组合物,其特征在于,包含权利要求1~3任一所述中药组合物的提取物以及药学上可接受的载体。
5.根据权利要求4所述的药物组合物,其特征在于,制剂形式包含颗粒剂、浸膏剂或合剂。
6.一种根据权利要求4或5所述的药物组合物的制备方法,其特征在于,包含以下步骤:
(1)按重量取各中药饮片提取两次;
(2)合并步骤(1)所得提取液,浓缩至60℃相对密度为1.08~1.12的浓缩液;
(3)当药物组合物为颗粒剂时,将步骤(2)所得浓缩液与糊精制粒。
7.根据权利要求6所述的制备方法,其特征在于,步骤(1)中两次提取的方法分别为第一次提取以中药饮片8~10倍质量的水,浸泡30~60min,煎煮40~60min,滤过;第二次提取以中药饮片6~10倍质量的水,煎煮30~60min,滤过。
8.根据权利要求6所述的制备方法,其特征在于,步骤(3)中制粒方法为进风温度:80~100℃,喷雾结束后60~70℃继续干燥。
9.根据权利要求1~3任一所述的中药组合物或者权利要求4或5所述的药物组合物在制备预防或/和治疗血管性认知障碍、混合型痴呆或伴有缺血症状的阿尔茨海默症药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述药物为预防或/和治疗肝肾不足、气虚血瘀、痰蒙神窍或大便秘结引发的痴呆的药物。
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