CN111973603A - neocaesalpin AH在制备治疗帕金森病的药物中的应用 - Google Patents
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Abstract
本发明属于药物技术领域,提供一种neocaesalpin AH的新应用,具体涉及neocaesalpin AH在制备治疗帕金森病的药物中的应用。本发明通过提取分离得到neocaesalpin AH,neocaesalpin AH可显著抑制秀丽隐杆线虫帕金森模型中α‑突触核蛋白的表达,对秀丽隐杆线虫帕金森病病理模型具有显著的治疗作用,提示neocaesalpin AH具有治疗帕金森病的潜力,可在制备治疗帕金森病的药物中应用,亦可在制备预防和辅助治疗帕金森病的保健品中应用。
Description
一、技术领域
本发明属于药物技术领域,提供一种neocaesalpin AH的新应用,具体涉及neocaesalpin AH在制备治疗帕金森病的药物中的应用。
二、背景技术
帕金森病(Parkinson’s disease,PD),又称震颤麻痹,是仅次于阿尔茨海默病的第二大神经退行性疾病。其临床特征主要表现为静止性震颤、肌肉强直、运动迟缓和姿势平衡障碍。黑质多巴胺能神经元的缺失导致黑质纹状体通路中多巴胺的减少和黑质神经元中出现蛋白聚集物(路易小体)是PD的两个病理特征,研究发现路易小体的主要成分是α-突触核蛋白。目前临床上对PD采用对症治疗的方式,主要以药物治疗为基础,这些药物是根据PD发病机制相关的关键靶点或信号通路开发的,仅可以用于缓解PD的症状,尚无有效的治疗方法恢复退化的神经元。
秀丽隐杆线虫(Caenorhabditis elegans)属于低等无脊椎动物,被生物学家们广泛用来进行基因组、细胞生物、细胞死亡、表观遗传以及抗衰老研究。它身体透明,有雌雄同体和雄虫2种性别特征,成虫体长1~2mm,以大肠杆菌为食,生命周期为3d。因其具有身体结构简单、生命周期短暂、繁殖迅速和易于培养等特点(赵亦周,2017),秀丽隐杆线虫在药物和靶蛋白筛选方面的研究中应用越来越普遍。
将人源α-突触核蛋白与YFP黄色荧光蛋白融合后插入线虫肌肉特异性启动子之下,使得人类α-突触核蛋白可在线虫肌肉组织特异性表达。α-突触核蛋白模型能够很好地模拟PD行为学改变、神经元结构、神经生化的改变过程,是研究PD发病机制、基因治疗以及药物干预的良好动物模型。线虫通体透明,利用荧光显微镜可清晰地观察到黄色荧光,荧光越强,α-突触核蛋白表达越高。对PD有治疗作用的药物可显著抑制α-突触核蛋白表达产生的荧光,待检药物抑制荧光能力越强,抗PD活性就越好(Fu et al.,2014)。
苦石莲(Caesalpinia minax)为豆科植物南蛇竻的种子,又名喙荚云实,老鸦枕头;其性苦寒,是一种药食同源植物,中医药用于散瘀,止痛,清热,去湿,治哕逆,痢疾,淋浊,尿血,跌打损伤。研究发现其具有抗炎、镇痛、抗肿瘤、抗肝损伤、降血脂、抗动脉粥样硬化等药理作用(江汉美,2013)。现有技术公开了neocaesalpin AH对MCF-7人癌细胞的体外杀伤活性(Ma et al.,2014)。并有报道neocaesalpin AH对核因子-κB(NF-κB)的表达有较强的抑制作用(Liu et al.,2020),但是对于neocaesalpin AH是否具有抗PD作用尚未可知。
基于上述研究现状,本发明公开了一种neocaesalpin AH的新应用,所述neocaesalpin AH可显著抑制α-突触核蛋白的表达,对秀丽隐杆线虫帕金森病病理模型具有显著的治疗作用,提示neocaesalpin AH具有治疗帕金森病的潜力,可在制备治疗帕金森病的药物中的应用,亦可在制备预防和辅助治疗帕金森病的保健品中应用。
三、发明内容
本发明的目的是提供一种neocaesalpin AH的应用。
具体涉及neocaesalpin AH在制备治疗帕金森病的药物中应用。
所述neocaesalpin AH作为药效成分的药物制剂为注射剂、散剂、颗粒剂、粉剂、丸剂、口服液、片剂等任一剂型,这是本领域技术人员可以理解的。
所述neocaesalpin AH亦可在制备预防和辅助治疗帕金森病的保健品中应用。
所述neocaesalpin AH作为有效成分的保健品剂型为散剂、颗粒剂、粉剂、丸剂、口服液、片剂等任一剂型,这是本领域技术人员可以理解的。
本发明所述neocaesalpin AH的制备方法如下:
称取苦石莲(Caesalpinia minax)(25kg),将其粉碎,室温下用95%乙醇浸泡提取四次,每次7天,所得乙醇部分减压蒸馏除去溶剂,得总浸膏1200g。将浸膏溶于1.5L蒸馏水中,之后用乙酸乙酯萃取三次,得到乙酸乙酯部分750g。将乙酸乙酯部分浸膏用硅胶柱层析分离,以石油醚-丙酮(40:1,20:1,10:1,5:1,2:1,1:1)为流动相梯度洗脱,之后通过薄层色谱实验(TLC)合并相似组分,最终得到6个组分(A-F)。
B组分(石油醚/丙酮20:1洗脱所得部分),63.0g,在大柱层析时得到大量的无色晶体,用石油醚/丙酮重结晶得到化合物neocaesalpinAH(图1)(1.3g)。
本发明以秀丽隐杆线虫PD病理模型进行实验,结果表明:neocaesalpin AH可显著抑制α-突触核蛋白的表达,对秀丽隐杆线虫PD病理模型具有显著的治疗作用,提示neocaesalpin AH具有治疗帕金森病的潜力,可在制备治疗帕金森病的药物中应用,亦可在制备预防和辅助治疗帕金森病的保健品中应用。
下面结合具体实施例进一步详细描述本发明的技术方案,但本发明的保护范围不局限于以下所述。
四、附图说明
图1neocaesalpin AH结构式。
图2本发明所述neocaesalpin AH减少OW13线虫α-突触核蛋白的积累。
C-空白对照组;P-阳性对照组。
五、具体实施方式
实施例1
本发明所述neocaesalpin AH的制备:
称取苦石莲(Caesalpinia minax)(25kg),将其粉碎,室温下用95%乙醇浸泡提取四次,每次7天,所得乙醇部分减压蒸馏除去溶剂,得总浸膏1200g。
将浸膏溶于1.5L蒸馏水中,之后用乙酸乙酯萃取三次,得到乙酸乙酯部分750g。将乙酸乙酯部分浸膏用硅胶柱层析分离,以石油醚-丙酮(40:1,20:1,10:1,5:1,2:1,1:1)为流动相梯度洗脱,之后通过薄层色谱实验(TLC)合并相似组分,最终得到6个组分(A-F)。
B组分(石油醚/丙酮20:1洗脱所得部分),63.0g,在大柱层析时得到大量的无色晶体,用石油醚/丙酮重结晶得到化合物neocaesalpin AH(图1)(1.3g)。
实施例2
本发明所述neocaesalpin AH对秀丽隐杆线虫PD病理模型的治疗作用:
1.生物材料
(1)秀丽隐杆线虫OW13(Punc-54:a-synuclein:YFP+unc-119)购自Caenorhabditis Genetics Center(CGC),为转基因品系。人类α-突触核蛋白基因与YFP融合蛋白在体壁肌肉中的特异性表达,用荧光显微镜观察,荧光越强,α-突触核蛋白表达越高。对PD有治疗作用的药物可显著抑制α-突触核蛋白表达产生的荧光,待检药物抑制荧光能力越强,抗PD活性就越好。本实施例采用秀丽隐杆线虫OW13株系作为筛选抗PD药物的病理模型。
(2)大肠杆菌OP50(尿嘧啶渗漏突变株),购自Caenorhabditis Genetics Center(CGC),作为秀丽隐杆线虫的食物。
2.试剂
(1)固体NGM(Nematode Growth Medium)培养基成分与制作:
成分 | 含量 |
NaCl | 3.00g |
K<sub>2</sub>HPO<sub>4</sub> | 2.34g |
KH<sub>2</sub>PO<sub>4</sub> | 17.23g |
蛋白胨 | 2.50g |
琼脂 | 17.00g |
补充H<sub>2</sub>O至 | 1000mL |
固体NGM培养基配制好后,121℃下高压恒温灭菌20min,冷却至80℃,在无菌操作台中加入5mg/mL胆固醇1mL,1MMgSO41mL,1MCaCl21mL摇匀,趁热倒入已灭菌的9cm培养板,约20mL/板。紫外照射20min,静置等待培养基凝固,每板加450μlOP50大肠杆菌菌液,涂匀晾干后置于37℃培养箱过夜培养。
(2)M9液配方:
成分 | 含量 |
Na<sub>2</sub>HPO<sub>4</sub> | 6.00g |
KH<sub>2</sub>PO<sub>4</sub> | 3.00g |
NaCl | 5.00g |
1MMgSO<sub>4</sub> | 1.00mL |
补充H<sub>2</sub>O至 | 1000mL |
(3)裂解液的配制:6.4%NaClO溶液和1M NaOH溶液按体积比1:1混合。
3.配制含有neocaesalpin AH的NGM平板
称取neocaesalpin AH0.0025g,加50μl DMSO溶解配成0.12M溶液,加入NGM培养基的终浓度分别为120μM、60μM和30μM,将NGM倒入各6cm培养皿,约10mL/板。紫外照射20min,静置等待培养基凝固,每板加200μl OP50大肠杆菌菌液,涂匀,晾干后置于37℃培养箱过夜培养。
4.实施步骤
(1)线虫的培养:
将秀丽隐杆线虫接在涂有大肠杆菌OP50的固体NGM板上,然后置于20℃的培养箱中培养,当线虫长到成虫时进行同步化处理。
(2)线虫同步化:
当NGM培养基含有大量成虫并且有部分虫卵已经孵出时,用M9缓冲液将线虫从培养基上洗下,转移到15ml离心管中,静置使线虫自由沉降至管底,弃去上清。向离心管中加入4ml裂解液,在涡旋搅拌器上振荡5-6分钟,线虫全部裂解后停止涡旋,分装于四个1.5mL离心管中,用M9溶液洗涤虫卵三次,后合并为两离心管置于20℃培养箱孵化30h。
(3)实验组设置
空白对照组:DMSO;
阳性对照组:50μM(n-butylidenephthalide);
neocaesalpin AH组:120μM;60μM;30μM。
(4)实验步骤
同步化后的虫卵孵化为L1期幼体线虫后,吸取10μl计数三次,取平均值,将线虫转移到含不同浓度的neocaesalpin AH的NGM培养基上,空白对照组为neocaesalpin AH组等体积DMSO的NGM培养基,阳性对照药为n-butylidenephthalide(Fuetal.,2014)。每个培养皿接300条线虫,每个药物浓度三个培养皿作为平行。20℃继续培养72小时后,用M9将线虫冲洗下来,4000rpm,5min离心除去上清(M9培养基),加入25mMNaN3麻醉线虫,在荧光显微镜下观察,拍照。每组随机观察40条线虫,所有图片采用ImageJ软件定量分析荧光强度。
(5)实验结果
实验结果如图2,纵坐标表示YFP荧光强度,其强度可以间接表示α-突触核蛋白在线虫体壁肌肉细胞中的表达量,此值越低,表明neocaesalpin AH抗PD活性越高,即neocaesalpin AH抗PD作用越强。星号(*)表示药物处理组和空白组之间具有统计学意义。
实验结果表明,在本实施例中neocaesalpin AH以剂量依赖方式降低OW13线虫YFP的表达,显著降低了人源α-突触核蛋白在线虫肌肉组织的含量,提示neocaesalpin AH对PD具有显著的治疗作用,最佳用药浓度为60μM。
通过以上实施例证明,neocaesalpin AH对秀丽隐杆线虫帕金森病病理模型具有显著的治疗作用,提示本发明所述neocaesalpin AH具有治疗帕金森病的潜力,可在制备治疗帕金森病的药物中应用,亦可在制备预防和辅助治疗帕金森病的保健品中应用。
Claims (5)
1.neocaesalpin AH在制备治疗帕金森病的药物中的应用。
2.如权利要求1所述的应用,其特征在于,所述neocaesalpin AH作为药效成分的药物制剂为注射剂、散剂、颗粒剂、粉剂、丸剂、口服液、片剂。
3.neocaesalpin AH在制备预防和辅助治疗帕金森病的保健品中的应用。
4.如权利要求3所述的应用,其特征在于,所述neocaesalpin AH作为有效成分的保健品剂型为散剂、颗粒剂、粉剂、丸剂、口服液、片剂。
5.如权利要求1或3任一项所述的应用,其特征在于,所述neocaesalpin AH的制备方法如下:
称取苦石莲(Caesalpinia minax)(25kg),将其粉碎,室温下用95%乙醇浸泡提取四次,每次7天,所得乙醇部分减压蒸馏除去溶剂,得总浸膏1200g。将浸膏溶于1.5L蒸馏水中,之后用乙酸乙酯萃取三次,得到乙酸乙酯部分750g。将乙酸乙酯部分浸膏用硅胶柱层析分离,以石油醚-丙酮(40:1,20:1,10:1,5:1,2:1,1:1)为流动相梯度洗脱,之后通过薄层色谱实验(TLC)合并相似组分,最终得到6个组分(A-F)。B组分(石油醚/丙酮20:1洗脱所得部分),63.0g,在大柱层析时得到大量的无色晶体,用石油醚/丙酮重结晶得到化合物neocaesalpin AH(图1)(1.3g)。
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CN110903268A (zh) * | 2019-12-19 | 2020-03-24 | 沈阳药科大学 | 沃卡帕烷型二萜衍生物及其制备方法和应用 |
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Non-Patent Citations (3)
Title |
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GUO-XU MA等: "Further Diterpenes from the Seeds of Caesalpinia minax Hance", 《HELVETICA CHIMICA ACTA》 * |
TING LIU等: "New cassane-type diterpenoids from kernels of Caesalpinia bonduc (Linn.) Roxb. and their inhibitory activities on phosphodiesterase (PDE) and nuclear factor-kappa B (NF-κB) expression", 《BIOORGANIC CHEMISTRY》 * |
陶丽珍等: "NF-κB通路与帕金森病", 《国际老年医学杂志》 * |
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