CN105267229A - 柴胡皂苷在制备调节多药耐药相关蛋白活性药物中的应用 - Google Patents

柴胡皂苷在制备调节多药耐药相关蛋白活性药物中的应用 Download PDF

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CN105267229A
CN105267229A CN201410230543.2A CN201410230543A CN105267229A CN 105267229 A CN105267229 A CN 105267229A CN 201410230543 A CN201410230543 A CN 201410230543A CN 105267229 A CN105267229 A CN 105267229A
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mrp
saikoside
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赵瑞芝
冯丽敏
陈振兴
王银洁
肖傅文
樊丹彩
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SECOND AFFILIATED HOSPITAL OF GUANGZHOU UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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SECOND AFFILIATED HOSPITAL OF GUANGZHOU UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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Abstract

本发明公开了柴胡皂苷在制备调节多药耐药相关蛋白活性药物中的应用，试验证明，所述的柴胡皂苷，可以调节MRP的活性，其对正常组织中的MRP的表达无影响，但可降低高表达组织中MRP的表达及活性，可在增强疗效的同时降低其毒副作用。本发明的MRP蛋白的抑制剂，可以调节MRP的活性，其对正常组织中的MRP的表达无影响，但可降低高表达组织中MRP的蛋白表达，增加其底物的摄取活性，可在增强疗效的同时降低其毒副作用，有利于逆转由于MRP活性导致的肿瘤多药耐药，能够满足临床应用的需要。

Description

柴胡皂苷在制备调节多药耐药相关蛋白活性药物中的应用
技术领域
本发明涉及柴胡皂苷的一种新的医药用途。
背景技术
多药耐药相关蛋白(MRP)是存在于机体多种组织细胞膜上，通过外排内源性及外源性毒物、信号传导分子等发挥防御功能与信息交换功能的蛋白，在肿瘤细胞如有高表达，是造成肿瘤多药耐药的原因之一，“Chen,Z.S.andTiwari,A.K.MultidrugResistanceProteins(MRPs/ABCCs)inCancerChemotherapyandGeneticDiseases,2011,FEBSJournal,278(18):3226-3245”文献公开了所述的MRP的相关信息和资料。而目前尚缺乏高效低毒的抑制剂。
发明内容
本发明的目的是提供一种柴胡皂苷在制备调节多药耐药相关蛋白活性药物中的应用，以满足人们的需要。
试验证明，所述的柴胡皂苷，可以调节MRP的活性，其对正常组织中的MRP的表达无影响，但可降低高表达组织中MRP的表达及活性，可在增强疗效的同时降低其毒副作用。
可以将治疗有效量的所述的柴胡皂苷与医药学上可接受的载体，采用本领域公知的方法，制备成为药物制剂，如乳剂、片剂、颗粒剂、口服液、胶囊、滴丸、丸剂、注射剂等，所述载体如香料、甜味剂、液体或固体填料或稀释剂等常用载体物质，制剂通常含有重量百分比为1-90％的有效成分，较佳重量含量为30-70％；
可以通过口服或注射途径施加于需要治疗的患者，剂量一般为10mg～50mg/体重/天，具体可根据患者的具体情况，由医师决定。
所述柴胡皂苷可采用商业化的产品，或者根据“柴胡皂苷a、c、d对照品的制备”文献报道的方法制备获得。
本发明的MRP蛋白的抑制剂，可以调节MRP的活性，其对正常组织中的MRP的表达无影响，但可降低高表达组织中MRP的表达及活性，可在增强疗效的同时降低其毒副作用，有利于逆转由于MRP活性导致的肿瘤多药耐药，能够满足临床应用的需要。
具体实施方式
实施例1
方法：
(1)摄取实验：分别采用给予谷胱甘肽(GSH)刺激和质粒转染的方法，得到高表达的MRP1-HEK293和MRP2-HEK293细胞；其中MRP1转运底物选用秋水仙碱、MRP2底物选用顺铂，以HPLC法分别测定各组细胞内底物含量，并以BCA法测定细胞总蛋白含量，计算单位蛋白摄取底物的含量。
具体操作过程：
1)HEK293细胞接种于100mm培养皿中，采用含GSH(2mM)的培养基进行培养，于对数生长期时加入相应的药物；细胞经药物处理48h后，移除药液；后加入秋水仙碱(50μmol·L-1)与细胞共孵育30min。随后收集细胞，预冷的PBS清洗3次，加入超纯水重悬细胞，-80℃冻融裂解；15000g离心15min，移取上清。
样品的处理以及细胞内秋水仙碱的含量测定方法参照文献“冯丽敏，张娴，赵瑞芝，醋柴胡对HEK293细胞中MRP1蛋白摄取活性、蛋白表达及mRNA水平的影响，辽宁中医杂志，2014，41(2)：310-312”
2)MRP2-HEK293细胞的构建：HEK293细胞接种于6孔板中，待细胞汇合度达80％时，每孔加入Opti-MEM培养基稀释的MRP2质粒(4μg/孔、4μg/孔和4μg/孔比例加入)，按质粒：转染试剂(W/V)＝1:3比例加入转染试剂MegaTran1.0室温孵育10min；反应10h后换液为完全培养基。细胞转染72h后，加入400μg·mL-1的G418溶液(氨基糖苷类抗生素)筛选8周。
MRP2-HEK293细胞接种于六孔板中，于对数生长期加入相应的药物处理24h后，再加入50μg·mL-1的顺铂溶液孵育4h；收集细胞、预冷PBS清洗，裂解细胞，通过HPLC法测定细胞内的顺铂含量。
MRP1的具体信息，可参见“ColeSP,DeeleyRG,TransportofglutathioneandglutathioneconjugatesbyMRP1,Trendsinpharmacologicalsciences2006,27(8):438-446”、“ElaineM.Leslie,RogerG.Deeley,SusanP.C.,Multidrugresistanceproteins:roleofP-glycoprotein,MRP1,MRP2,andBCRP(ABCG2)intissuedefense,ToxicologyandAppliedPharmacology2005,204(3):216-237”
MRP2的具体信息，可参见“CCPaulusma,MKool,PJBosma,GLScheffer,FterBorg,RJScheper,GNTytgat,PBorst,FBaasandROElferink,AmutationinthehumancanalicularmultispecificorganicaniontransportergenecausestheDubin-JohnsonsyndromeHepatology1997,25(6):1539–1542”,“TPSchaub,JKartenbeck,JOVogel,R.Witzgall,WkrizandD.Keppler,Expressionoftheconjugateexportpumpencodedbythemrp2geneintheapicalmembraneofkidneyproximaltubules,JournaloftheAmericanSocietyofNephrology,1997:1213-1221”、“AldoD.Mottino,TimHoffman,LotharJennes,JingsongCao,MaryVoreExpressionofmultidrugresistance-associatedprotein2insmallintestinefrompregnantandpostpartumrats,AmericanJournalofPhysiology-GastrointestinalandLiverPhysiology,2001.280:G1261-G1273”.
HEK293的具体信息，可从美国菌种典藏中心(ATCC)获得，且附有详细介绍和说明。
(2)以WesternBlot法测定各组细胞中MRP1或MRP2相对蛋白表达量。
结果：
以高表达细胞组摄取量和蛋白表达量为100％计算，柴胡皂苷对各组细胞中底物摄取的影响见表1，对蛋白影响的结果见表2。
表1柴胡皂苷对MRP1及MRP2底物摄取的影响
不加柴胡皂苷(％) 加柴胡皂苷(％)
正常细胞株 100 92
MRP1过表达细胞株 100 152
MRP2过表达细胞株 100 158
表2柴胡皂苷对MRP1及MRP2蛋白表达的影响
不加柴胡皂苷(％) 加柴胡皂苷(％) P值
正常细胞株 100 95±3 >0.05
MRP1过表达细胞株 100 22.2±2.3 <0.01
MRP2过表达细胞株 100 36.2±3.2 <0.01
从上表可以看出，柴胡皂苷不影响正常细胞MRP1及MRP2的蛋白表达以及底物摄取；但显著减低过表达细胞株中这两种蛋白的表达，并增加其底物摄取，提示其作为肿瘤辅助治疗用药可增强疗效且具有较低的毒性。

Claims (6)

1.柴胡皂苷在制备调节多药耐药相关蛋白活性药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述药物，包括治疗有效量的柴胡皂苷与医药学上可接受的载体。
3.根据权利要求2所述的应用，其特征在于，所述药物为乳剂、片剂、胶囊、颗粒剂、口服液、滴丸、丸剂或注射剂。
4.根据权利要求2所述的应用，其特征在于，所述制剂含有重量百分比为1-90％的有效成分。
5.根据权利要求1所述的应用，其特征在于，通过口服或注射途径施加于需要治疗的患者。
6.根据权利要求5所述的应用，其特征在于，剂量为20mg～50mg/体重/天。
CN201410230543.2A 2014-05-28 2014-05-28 柴胡皂苷在制备调节多药耐药相关蛋白活性药物中的应用 Pending CN105267229A (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
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JPH0967388A (ja) * 1995-08-31 1997-03-11 Shiseido Co Ltd サイコサポニンの製造方法
CN102659903A (zh) * 2012-05-10 2012-09-12 青川德康源药业有限公司 柴胡皂苷a或柴胡皂苷d的提取纯化方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0967388A (ja) * 1995-08-31 1997-03-11 Shiseido Co Ltd サイコサポニンの製造方法
CN102659903A (zh) * 2012-05-10 2012-09-12 青川德康源药业有限公司 柴胡皂苷a或柴胡皂苷d的提取纯化方法

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