CN102188477B - 龙胆提取物活性组分的制备及应用 - Google Patents
龙胆提取物活性组分的制备及应用 Download PDFInfo
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Abstract
本发明提供一种从中药龙胆中提取的龙胆活性组分,呈白色,有较刺激性气味,易溶于氯仿,溶于正己烷,微溶于乙醇和甲醇,用TLC分析时,以氯仿为展开剂,在高为5cm正向Silica板上,其Rf值约为0.4,该样品在248nm和320nm波长下具有显著的紫外吸收,该活性组分在龙胆干燥根及根茎中的含量约为0.1%。本发明的龙胆活性组分通过有机溶剂浸提、溶剂分配、开口色谱柱分离得到,具有显著类似神经生长因子活性,能促进成年脑的神经元新生,阻止β-淀粉肽的神经损伤和抑制神经再生的作用,可在制备治疗缺血性脑损伤和防治老年性痴呆等神经退行性疾病药物中的应用。
Description
技术领域
本发明属于中药的药物用途领域,具体涉及一种以中药材龙胆为原料制备提取物活性组分及其在制备治疗缺血性脑损伤和防治老年性痴呆等神经退行性疾病药物中的应用。
背景技术
老年性痴呆症主要分为二大类型:阿尔茨海默症(Alzheimer’s disease, 简称AD)和血管性痴呆症。(1)脑缺血性疾病是临床最常见的疾病之一,具有发病率、致残率和病死率高的特点,严重威胁着人类的健康。随着全世界人口逐步老龄化,缺血性脑损伤的发病有逐年增加趋势,但是迄今在国内外仍然缺乏有效的预防和治疗措施。中枢神经细胞对缺氧伤害的高敏感性和缺乏再生能力的特征被认为是导致脑卒中后遗症——血管性痴呆症的主要原因。因此,如何阻止或减轻脑缺血和再灌流后的神经细胞死亡一直是神经科学研究和神经疾病药理学研究的焦点。(2)阿尔茨海默症是一种神经退行性疾病,以记忆力和认知功能损害为主要临床病症,严重时会导致生活不能自理。目前阿尔茨海默症已成为成年人死亡的第四大病因,仅次于心脏病、癌症、中风。我国阿尔茨海默症患者已超过500万, 约占世界总病例数的1/4;而且,随着人口老龄化的加快,这个数字将更为庞大,给社会稳定与发展带来重大的影响。因此,开发有效的预防治疗老年性痴呆等神经退行性疾病的药物已成为全世界迫切需要解决的医学问题。
现阶段治疗老年性痴呆的药物种类甚多,主要有胆碱能药,其中乙酰胆碱酯酶(Acetylcholinesterase, AChE)抑制剂,主要上市的药物有他克林(tacrine)、酒石酸卡巴拉汀(rivastigmine)、石杉碱甲(huperzine A)、多奈哌齐(donepezil)等;β、γ分泌酶抑制药;脑代谢调节剂,如长春胺、尼莫地平、脑益嗪;影响自由基代谢的药物,如维生素C结合维生素E等。但这些治疗药物只能延缓AD病程的进展,且随病情发展药物疗效逐渐降低,有副作用,故人们将目光转向新的抗老年痴呆药物的研发,寻找针对AD病因的治疗药物和方法,成为当今研究的热点和难点。
研究表明,神经营养因子对神经发育和成年神经系统的疾病过程都有重要的影响。在神经退行性变动物模型中,发现神经生长因子(nerve growth factor,NGF)能阻止或减少神经元的退变。NGF是人类发现的第一个最重要的神经营养因子,它对神经细胞的生长、发育、分化和功能保持等方面有重要调控作用的生物活性多肽。研究发现,神经营养因子能阻止或减少神经萎缩、神经变性,促进外伤后的神经修复。然而,神经营养因子是一个由100多个氨基酸组成的蛋白质;由于分子量大和极性强等原因,不能通过血脑屏障,是限制神经生长因子临床应用的瓶颈。因此,寻找具有类似NGF活性(NGF mimicr)或能增强其活性(NGF enhancer)并且能通过血脑屏障的低分子化合物就自然成为了研究热点。
中草药是中药学的物质基础,是天然活性有机化合物的聚宝盆。我国有着几千年的中医文明,且地大物博,有很多宝贵的道地药材,因为中药材资源的利用较方便,所以对其新的成分和新的活性的研究也就十分重要。中药对老年性痴呆症有显著疗效,其中最著名的是从蛇足石杉中提取的生物碱-石杉碱甲(huperzine A),它是中国科学院药物研究所开发的具有自主知识产权的创新药物;它不仅是一种高效高选择性的乙酰胆碱酯酶(AChE)抑制药,而且还能减少谷氨酸诱发的神经细胞死亡;具有明显的保护神经细胞对抗β淀粉样多肽(Aβ)产生的氧化应激反应;能够对抗过氧化氢、蛋白激酶C抑制剂等诱导的神经细胞凋亡作用。
龙胆,别名:苦胆草、胆草。春、秋二季采挖,洗净,干燥。性味,苦,寒。归肝、胆经。2005版药典中介绍,龙胆的功能主治:清热燥湿、泻肝胆火。用于湿热黄疸、阴肿阴痒、带下、强中、湿疹瘙痒、目赤、耳聋、胁痛、口苦、惊风抽搐。迄今为止,未见关于龙胆活性组分有类似神经生长因子活性的报道。
发明内容
本发明提供一种具有显著类似神经生长因子活性的龙胆活性组分(n-GS),呈白色,有较刺激性气味,易溶于氯仿,溶于正己烷,微溶于乙醇和甲醇。用TLC分析时,以氯仿为展开剂,在高为5cm正向Silica板上,其Rf 值的范围是0.3-0.5,该样品在248 nm和320nm波长下具有显著的紫外吸收,该活性组分在龙胆干燥根及根茎中的含量范围是0.08%-0.1%。
本发明的另一个目的是提供所述的龙胆活性组分n-GS的制备方法,通过以下步骤实现:
1)粉碎和浸提:
中药龙胆粉碎后,用乙醇(药用级)室温下浸提4-5天(偶尔震荡),抽滤浓缩,得乙醇浸提物粗样,再用80%甲醇水溶液和正己烷萃取分离,得到正己烷层粗样;
2)分离和纯化:
将正己烷层粗样经一次硅胶开口柱分离(200-300目,溶剂系统为正己烷:氯仿),收集合并正己烷:氯仿(7:3)的洗脱部位、浓缩,得到具有显著的NGF mimics的龙胆活性组分。
本发明在PC12细胞生物活性鉴定系统的引导,经过简单提取分离,从坚龙胆Gentiana rigescens Franch. 的干燥根及根茎中获得一种活性组分,其具有显著类似神经生长因子活性,根据Gentisides命名为n-GS。
本发明的再一个目的是提供该活性组分n-GS在制备治疗缺血性脑损伤和防治老年性痴呆等神经退行性疾病药物中的应用。以及在制备防治老年性痴呆等神经退行性疾病的食品中的应用。所述药物和食品含有药学或食品可接受的载体或稀释剂。
所述的药学上可接受的载体是指药学领域常规的药物载体,例如稀释剂、赋形剂如是等,填充剂如淀粉、蔗糖、微晶纤维素等;粘合剂如淀粉浆、羟丙纤维素、明胶、聚乙二醇等;湿润剂如硬脂酸镁、微粉硅胶、聚乙二醇类等;吸收促进剂聚山梨脂、卵磷脂等,表面活性剂伯洛沙姆、脂肪酸山梨坦、聚山梨脂等等,另外还可以在组合物中加入其它辅剂如香味剂、甜味剂等。
本发明所述的活性组分n-GS可以以单位剂量形式给药,给药途径可为肠道和非肠道,包括口服、肌肉、皮下和鼻腔。
本发明所述的活性组分给药途径可为静脉给药。注射包括静脉注射、肌肉注射、皮下注射和穴位注射。
本发明的药物组合物的各种剂型可以按照药学领域的常规生产方法制备,例如使活性组分与一种或多种载体混合,然后将其制成所需的剂型。
给药剂型可以是固体制剂、胶囊剂或液体制剂,包括片剂、胶囊剂、分散片、口服液、大输液、小针、冻干粉针、软膏、搽剂或栓剂。
本发明的有益效果在于:
(1)有机溶剂浸提、溶剂分配、开口色谱柱分离得到,制备方法具有简单、快速等优点;
(2)动物药理学实验结果证明,中药龙胆中分离纯化得到的活性组分——n-GS口服或腹腔给药能快速通过血脑屏障,对脑卒中具有显著治疗作用,具体的说,n-GS腹腔注射能透过血脑屏障,减小脑缺血导致的脑梗塞面积和减轻脑水肿,并减少神经细胞死亡;
(3)n-GS具有显著的类似神经生长因子的活性,促进成年脑的神经元新生,阻止β-淀粉肽的神经损伤和抑制神经再生的作用,具有预防和治疗老年性痴呆症的作用。
本发明开拓了n-GS新的药物用途,为预防及治疗缺血性脑损伤和老年痴呆症等神经退行性疾病提供新的保健食品和治疗药物。
附图说明
图1是加入n-GS,经48小时后PC 12细胞的神经突起分化率随剂量增加的变化。
图2是加入n-GS,经48小时后PC 12细胞神经突起的显微照片。
图3是n-GS对缺血性脑损伤有浓度依赖性的保护作用。
图4是n-GS保护缺血性脑损伤有24小时以上的有效时间窗。
图5是n-GS保护缺血性脑损伤能改善脑卒中后运动障碍。
图6A-6D是n-GS能促进成年脑神经再生。
图7A-7B是n-GS具有阻止Aβ的神经毒作用,改善Aβ小鼠的认知障碍。
图8A-8E是n-GS改善APP/PS1小鼠的海马神经再生障碍。
具体实施方式
以下通过对该活性组分制备实例的实施方式和附图再对本发明的上述内容作进一步的详细说明,但不应将此理解为本发明上述主题的范围仅限于下述的实例,凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1 n-GS的制备
从中药龙胆中获得具有显著的NGF mimic活性的活性组分n-GS的制备方法,具体步骤为:
1)粉碎和浸提:
1000 g中药龙胆粉碎后,用5 L乙醇(药用级)室温下浸提5天(偶尔震荡)。抽滤浓缩后,得到乙醇浸提物粗样138.98 g,用80%甲醇水溶液(750 ml)和正己烷交替萃取分离,直至正己烷层澄清。共得到正己烷层粗样13 g。
2)分离和纯化:
将正己烷层粗样经一次硅胶开口柱分离(200-300目,以下溶剂系统均按体积比,正己烷:氯仿 = 100:0, 90:10, 80: 20, 70:30, 50:50),所需活性组分主要在正己烷:氯仿(7:3)的洗脱部位,合并浓缩该部位,得到活性组分n-GS 905 mg 。
经上述方法制备得的活性组分,呈白色,有较刺激性气味,易溶于氯仿,溶于正己烷,微溶于乙醇和甲醇。用TLC分析时,以氯仿为展开剂,在高为5cm正向Silica板上,其Rf值约为0.4,该样品在248 nm和320 nm波长下具有显著的紫外吸收,龙胆中的含量约为0.1%。
实验方法:
1)PC 12细胞的培养:接20×104个PC 12细胞于100 mm的培养皿中,含10 ml DMEM培养基(其中含10%马血清、5%胎牛血清),两天后更换一次培养基,再过三天继代。先用PBS将细胞洗两次,再加入10 ml PBS于培养皿中,在37 ℃,5% CO2的培养箱内培养10分钟,吹洗,转移到15 ml的一次性离心管,离心后血球计数板上计数。24孔细胞培养板每孔先加入1 ml含血清的DMEM培养基,细胞计数后,每孔接2×104个细胞,CO2培养箱培养24小时后加样。
2)活性测试:以DMSO为阴性对照,NGF 40 ng为阳性对照,将n-GS配置成不同浓度的DMSO溶液。用1 ml含1% DMSO和样品的DMEM溶液(不含血清)将24孔细胞板的每孔原培养基取代后,放入37 ℃,5% CO2的培养箱中培养。倒置显微镜下每隔24小时、连续6天观察细胞形态变化,记录细胞的神经突起分化率 (神经突起长于胞体直径一倍的细胞数目与视野下总细胞数目的比值),每个视野下约100个细胞,随机选取3处,并统计作图分析。
3)实验结果:
参见图 1、图2,在1-30 μg/ml的浓度下,48小时候后活性组分n-GS显示出很好的NGF-mimics活性。图 1中C:阴性对照1% DMSO;NGF (40ng/ml):阳性对照)。图 2中a, 1% DMSO为阴性对照;b, NGF 40 ng/mL,为阳性对照;c, 活性组分n-GS (3g/ml);d, 活性组分n-GS (10 g/ml)。
实施例2 n-GS对缺血性脑损伤有保护作用,并能部分改善脑卒中后运动障碍
实验主要材料:昆明小鼠(雄性和雌性各40只),体重25 g~30 g,购自江苏省实验动物中心。
实验操作:10 %水合氯醛腹腔麻醉,结扎右侧颈总动脉与颈外动脉,用动脉夹夹闭颈内动脉远心端,将渔线插入颈内动脉远端,实施大脑中动脉60分钟阻塞(大脑中动脉阻塞在图中简称MCAO),制备局灶性脑缺血小鼠模型(用电热毯以保持实验动物肛门温度在37±0.5度)。在脑缺血后1小时按每公斤体重0.5、1、5、10、20 mg剂量进行n-GS腹腔注射,共给药两次(间隔8小时)。或在脑缺血后1、6、8、12、24小时按每公斤体重10mg剂量进行n-GS腹腔注射,共给药两次(间隔8小时)。脑缺血后48小时取出全脑,放置-20度冷冻10分钟,做全脑2毫米厚冠状连续切片。用2% TTC染色后,进行image-J 软件处理图像分别测出每张脑片梗死区及非梗死区的面积。计算脑梗死体积的百分比:(梗死区体积平均值/非缺血侧大脑半球体积平均值)×100%。脑缺血后28天,进行矿场实验,检测脑缺血后小鼠的运动功能。各实验组均是8只小鼠。
实验结果:60分钟的大脑中动脉阻塞引起大约36.4%的脑梗死(**P<0.01,图3)。n-GS两次给药能浓度依赖性地减少缺血后脑梗死的体积(*P<0.05,**P<0.01,图3),提示n-GS治疗能减轻缺血后脑梗死。n-GS的神经保护作用呈现长达24小时的有效时间窗(图4)。n-GS治疗能部分地恢复脑缺血后小鼠的运动功能障碍,改善脑缺血造成的行走距离(**P<0.01与对照组相比,# P<0.05与脑缺血组相比,图5A)和站立次数的降低(** P<0.01与对照组相比,# P<0.05与脑缺血组相比,图5B),提示n-GS是一种很有潜力的治疗脑卒中的新化合物。
实施例3 n-GS促进成年的神经新生
用BrdU(5-溴脱氧尿核苷)标记有丝分裂期细胞,发现成体哺乳动物海马的神经干细胞能分化为神经细胞——神经再生。新生神经元能与CA3区神经元建立突触联系和发生突触可塑性。认为成年脑的神经再生可以取代自然老化或病变死亡的神经元,维护脑功能和结构的完整性。本实验观察PS对神经再生过程(主要包括神经干细胞增殖、存活、分化)的影响。
实验主要材料:2月龄昆明小鼠(雄性和雌性各40只),体重25g~30g,购自江苏省实验动物中心。
实验操作:BrdU连续注射三次,间隔8小时。n-GS给药方案参见图6A的上部。在BrdU给药后1、10或28天用免疫组化的方法检测海马DG区的BrdU阳性(BrdU+)细胞,以确定n-GS对细胞增殖(24小时龄-BrdU+细胞)、存活(10天龄-BrdU+细胞)和成熟(28天龄-BrdU+细胞)的影响。用Doublecortin(DCX)免疫染色,观察n-GS对新生神经元突起生长的影响。
实验结果(图6): n-GS能促进神经干细胞的增殖(图6A)和存活(图6B-C),促进新生神经元的突起生长(图6D),提示n-GS具有神经营养作用。
实施例4 n-GS具有阻止Aβ的神经毒作用
实验主要材料:昆明小鼠(雄性和雌性各40只),体重25 g~30 g,购自江苏省实验动物中心。
实验操作:采用立体定位技术,将Aβ(25-35)注射进入侧脑室(前囟点后0.3mm,右侧1mm,深2.5mm)。Aβ(25-35)注射后当天开始进行连续7天的n-GS(10mg/kg)腹腔注射。在Aβ(25-35)注射后第3天进行水迷宫测试,记录大鼠的运动轨迹,计算登台潜伏期。在注射后7天,用4%多聚甲醛经左心室灌注固定脑组织。石蜡包埋后,做海马连续切片。1%甲苯胺蓝染色后,观察海马CA1区存活锥体细胞。
实验结果(图7):Aβ引起大约60%的海马CA1区神经元死亡(图7A),逃避潜伏期延长(图7B),提示空间认知功能降低。n-GS处理能减少海马CA1区神经元死亡,改善空间认知功能降低。
实施例5 n-GS具有改善AD脑神经再生障碍的作用
阿尔茨海默病(Alzheimer’s disease, AD)是一种以进行性认知功能障碍为特征的神经系统退行性疾病。新生神经元是否能替代病变的神经元,改善AD的认知功能障碍已成为AD研究的一个新靶点。研究发现,AD脑海马的前体细胞向神经元的分化比例明显减少,同时新生神经元的存活率显著降低,并且新生神经元的突起生长异常。这些研究都已证实,AD脑的神经再生过程受到严重的损害,提示神经再生障碍可能是AD认知功能进行性减退的重要病理机制之一。
实验主要材料:APP/PS1转基因AD小鼠(10月龄雌雄鼠)(购于南京大学动物模式中心)。
实验操作:用BrdU(5-溴脱氧尿嘧啶核苷)连接12天皮下注射,标记有丝分裂期的细胞,从BrdU注射前15天到BrdU注射后第28天进行n-GS口服或腹腔给药(5和10mg/kg)。然后在BrdU末次给药后28天用免疫组化的方法检测海马DG区的BrdU阳性(BrdU+)细胞,观察n-GS对新生神经存活的影响。通过DCX染色能观察新生神经细胞突起的生长。
实验结果(图8):与野生型小鼠相比,APP/PS1小鼠齿状回的28天龄-BrdU+细胞显示50%的减少(图8A)。n-GS治疗能改善APP/PS1小鼠28天龄-BrdU+细胞的减少(图8B)。此外,APP/PS1小鼠新生神经元突起的长度比野生型小鼠明显降低(图8C),而n-GS治疗能保护APP/PS1小鼠的神经突起生长(图8D)。n-GS治疗能部分改善APP/PS1小鼠的空间认知功能(图8E),提示n-GS促进AD脑神经元再生对认知功能障碍有改善作用。
实施例6 n-GS灌胃给药未出现毒性反应
急性中毒试验
实验主要材料:ICR小鼠(体重22g~25g),雄性和雌性各60只,购自浙江大学实验动物中心。雌雄各半,随机分为对照组,n-GS处理组。
实验操作:n-GS较难溶于水。将n-GS溶解于99.5%乙醇中,然后添加1%吐温80,用生理盐水稀释到使用浓度(乙醇的最终浓度小于2%)。经口服药n-GS为5g/kg。连续14天观察动物的精神状态,测定体重及摄食量。小鼠无死亡情况,摄食量无明显变化,但体重变化明显减少。心,肝,脾,肾等组织重量及眼观无显著性差异。
实验结果:与生理盐水对照组相比,n-GS处理组动物没有出现体重增加/减少(包括各脏器重量),也没有出现呼吸(50~60次/分钟)、心率(305~400次/分钟)、平均动脉压(210mmHg)的异常,以及昏睡、狂躁、运动行为异常等现象。表1显示,心,肝,脾,肾等组织重量。
慢性毒性试验
实验主要材料:SD大鼠(体重170g~240g),雄性和雌性各60只,购自浙江大学实验动物中心。雌雄各半,随机分为对照组,n-GS处理组。
n-GS连续90天的腹腔注射(10 mg/kg/天),或经口给药(10 mg/kg/天)(生理盐水给药作为对照组)后,检测体重。用10%水合氯醛腹腔麻醉,记录呼吸、心率和平均动脉压。然后从左心室取血进行血液学和血液生化学指标等的检测。并取出各内脏器官进行组织学的观测。
实验结果:与生理盐水对照组相比,n-GS处理组动物没有出现体重和各脏器重量的增加/减少(表2)。
Claims (1)
1.一种龙胆提取物活性组分的制备方法,其特作在于,通过以下步骤实现:
(1)粉碎和浸提:
中药龙胆粉碎后,用药用级乙醇室温下浸提4~5天,抽滤浓缩,得乙醇浸提物粗样,再用80%甲醇水溶液和正己烷萃取分离,得到正己烷层粗样;
(2)分离和纯化:
将正己烷层粗样经一次硅胶开口柱分离, 200-300目,溶剂系统为正己烷:氯仿,收集合并正己烷:氯仿=7:3的洗脱部位,浓缩,得到龙胆活性组分;
所述龙胆提取物活性组分呈白色,有较刺激性气味,易溶于氯仿,溶于正己烷,微溶于乙醇和甲醇,用TLC分析时,以氯仿为展开剂,在高为5cm正向Silica板上,其Rf 值是0.3-0.5,该样品在248 nm和320nm波长下具有显著的紫外吸收,龙胆中的含量是0.08%-0.1%。
2.根据权利要求1所述方法获得的一种龙胆提取物活性组分在制备治疗缺血性脑损伤和防治老年性痴呆疾病药物中的应用。
3.根据权利要求1所述方法获得的一种龙胆提取物活性组分在制备防治老年性痴呆食品中的应用。
4.根据权利要求2或3所述的应用,其特征在于,所述药物或食品的剂型为固体或液体。
5.根据权利要求2所述的应用,其特征在于,所述药物的给药途径为肠道或非肠道。
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