CN106467564A - 一种谷精草酚酸类富集流分及在制备抗肿瘤药物中的应用 - Google Patents
一种谷精草酚酸类富集流分及在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明一种谷精草酚酸类富集流分及在制备抗肿瘤药物中的应用,属于制药领域,具体涉及一种谷精草酚酸类富集流分、其制备方法及在制备具有极光激酶A/B抑制作用药物中的应用。采用细胞生物学方法测定了谷精草中酚酸类富集流分对人白血病细胞(K562)的抑制增殖活性及机制,结果表明谷精草中酚酸类成分是通过抑制极光激酶A/B的活性达到抑制细胞增殖作用的,谷精草作为一种药食两用的中草药,有望开发为一种高效低毒的抗肿瘤药物应用于临床。
Description
技术领域:
本发明属于制药领域,具体涉及一种谷精草酚酸类富集流分、其制备方法及在制备具有极光激酶A/B抑制作用药物中的应用。
背景技术:
极光激酶家族是细胞有丝分裂调控网络中的一类重要的丝氨酸/苏氨酸激酶,包括极光激酶A、B、C三个家族成员。在细胞有丝分裂中,极光激酶参与了中心体成熟分离、染色体固缩、纺锤体组装和维持、染色体分离以及胞质分裂等多个过程,自1995年发现、1998年首次观察到其在人类肿瘤组织表达后,极光激酶逐渐成为学术界及肿瘤学界的研究热点协同高效地抑制恶性肿瘤细胞生长。
极光激酶家族成员中的A和B在肿瘤组织中的表达水平呈现同步的升高,即极光激酶B上调表达往往同时伴有极光激酶A水平的上调,进一步的研究明确了极光激酶A和B在乳腺癌、结直肠癌、前列腺癌、肺癌、神经胶质瘤及白血病等中均有过表达现象,目前认为极光激酶A可以改变表达细胞携带的遗传缺陷,其次,极光激酶B过表达诱导肿瘤转移。
极光激酶A/B在肿瘤组织中特异性过表达并参与肿瘤细胞有丝分裂的各个阶段,与肿瘤的发生和发展紧密相关,是当前极具潜力的肿瘤治疗靶点。因此,寻找高效低毒的极光激酶A/B抑制剂成为抗肿瘤药物研究的热点。
谷精草(Eriocaulon buergerianum)又名谷精珠、珍珠草,为中医常用中药。昧甘,性平,归肝、胃经,可疏散风热,明目退翳。研究表明谷精草中含有谷精草素、生物碱、酚酸类成分等化学成分。主要的药理作用有抗肿瘤、抑菌、抗氧化等。谷精草也常常被做成养生保健茶,具有清热明目等功效。虽然根据药学大辞典记载,谷精草的水煎剂有抗肿瘤活性,但其作用机制以及活性成分均不明确。目前也没有谷精草中酚酸类成分在抑制极光激酶A/B方面的报道。
发明内容:
发明目的:本发明提供了谷精草中酚酸类富集流分对极光激酶A/B的抑制活性,旨在将酚酸类化合物继续研究开发得到较好的具有极光激酶A/B抑制活性的新型药物。
技术方案:
一种谷精草酚酸类富集流分,其特征在于:从谷精草中分离出酚酸类富集流分具有活性的化合物如下:
一种如上所述谷精草酚酸类富集流分的制备方法,其特征在于:取谷精草药材10kg,用60%乙醇回流提取3次,每次2小时,回收乙醇,得浸膏;将浸膏分散于水中,以石油醚、二氯甲烷、乙酸乙酯、正丁醇萃取;将乙酸乙酯萃取物经HPD101大孔树脂分离,依次以水E0,10%的乙醇-水E1、30%乙醇-水E2、70%乙醇-水E3以及95%乙醇E4进行洗脱;其中的70%流分为E3;以没食子算为对照品,并采用HPLC法测定了E3中总酚酸的含量17.85%;E3经过反复硅胶色谱、Sephadex LH-20、ODS及制备液相分离手段,并结合理化性质和现代波谱学手段从中分离鉴定出酚酸类单体化合物。
一种谷精草酚酸类富集流分在制备抗肿瘤药物中的应用。
谷精草酚酸类富集流分对人白血病细胞(K562)的具有抑制增殖活性且作用机制是通过抑制极光激酶A/B的活性,进而引起细胞周期阻滞、诱导细胞凋亡。
优点及效果:本发明采用MTT测定谷精草中酚酸类富集流分对K562细胞的增殖抑制作用,流式细胞术分析对细胞周期及细胞凋亡的影响,并应用Western blot方法检测对相关蛋白表达的影响。结果表明谷精草中酚酸类富集流分对K562细胞体现良好的抑制增殖的作用,并产生了明显的G2/M期细胞周期阻滞,同时能下调极光激酶的特异性底物Histone H3(Ser10)的磷酸化水平,表明谷精草中酚酸类富集流分能够有效的抑制极光激酶的活性。此外,Aurora A(Thr288)与Aurora B(Thr232)的磷酸化水平也被谷精草的酚酸类富集流分所抑制,进一步确证谷精草的酚酸类富集流分是一种有效的极光激酶抑制剂。
本发明采用生物学方法测定了谷精草中酚酸类富集流分对人白血病细胞(K562)的抑制增殖活性及机制,结果表明谷精草中酚酸类富集流分是通过抑制极光激酶A/B的活性达到抑制细胞增殖作用的。因此本研究发掘了谷精草中酚酸类成分的药用前景,开拓了一个新的药用领域。
附图说明:
1、附图1为本发明对肿瘤细胞生长的量效关系图;
2、附图2为本发明对K562细胞产生了明显的G2/M期阻滞图;
3、附图3为本发明促进Annexin V阳性细胞率明显大幅度增加效果图;
4、附图4为本发明对极光激酶活性的电化学发光免疫分析图。
具体实施方式:
下面结合实施例和附图对本发明作进一步的描述。
取谷精草药材(约10kg),用60%乙醇回流提取3次,每次2小时,回收乙醇,得浸膏。将浸膏分散于水中,分别以石油醚、二氯甲烷、乙酸乙酯、正丁醇萃取。经检测,以乙酸乙酯萃取物为最具活性。乙酸乙酯萃取物经大孔树脂(HPD101大孔树脂)分离,依次以水(E0),10%的乙醇-水(E1)、30%乙醇-水(E2)、70%乙醇-水(E3)以及95%乙醇(E4)进行洗脱。其中的70%流分为Fraction 3(E3)。以没食子算为对照品,并采用HPLC法测定了E3中总酚酸的含量17.85%。E3经过反复硅胶色谱、Sephadex LH-20、ODS及制备液相分离手段,并结合理化性质和现代波谱学手段从中分离鉴定出酚酸类单体化合物的结构如下:
实施例2:谷精草中酚酸类富集流分体外抗肿瘤机制研究。
(1)仪器与试剂:RPMI Medium 1640培养液、胎牛血清购自美国Hyclone公司。Trypan blue、Tris-base、丙稀酰胺、亚甲双丙稀酰胺、十二烷基素硫酸钠(SDS)、四已基已二胺(TEMED)、过硫酸钠、二甲基二苯基四氮唑溴盐(MTT)购自美国Amresco公司。甘氨酸、碘化丙啶(PI)购自美国Sigma公司。Annexin V-FITC assay kit购自凯基生物公司。CKX31型倒置显微镜(菲律宾奥林巴斯公司);恒温CO2培养箱(美国Thermo公司);5810R型台式高速低温离心机(德国Eppendorf公司);全自动酶标仪(美国Biotek集团)。
(2)细胞培养:K562培养于1640培养基(含10%胎牛血清),37℃、含5%CO2湿化恒温孵箱中培养。每2-3天传代一次。实验时取对数生长期细胞,0.4%台盼蓝鉴定细胞活性在98%以上。
(3)细胞生长抑制测定:取对数生长期的K562细胞,调整密度至1×105个/mL,接种于96孔板,100μL/孔,培养1h后分别加入不同浓度的谷精草萃取层及酚酸类富集流分E3,每个浓度设3个复孔,在37℃、5%CO2孵箱培养24h后,每孔加5g/L的MTT 20μL继续孵育4h,然后加三联液,每孔加入100μL,继续培养12~16h后,振荡5min,用酶标仪测定OD560nm值,以OD560nm间接反映存活细胞数量。据此可推测各浓度药物对细胞的抑制率。抑制率%=(OD对照组-OD加药组)/OD对照组×100%各组实验均重复3次,实验数据用SPSS 17.0统计分析。
结果如下:谷精草体外抗肿瘤活性实验表明:谷精草的石油醚、二氯甲烷、乙酸乙酯及正丁醇提取物对K562均有一定的增殖抑制作用,乙酸乙酯层的抗肿瘤活性较好。经过处理后得到的酚酸类富集流分E3抑制活性增强,并随着药物浓度的增加,药物对细胞生长的抑制作用增加,药物对肿瘤细胞的生长具有很好的量效关系(图1中的A、B)。注:PE、DCM、EAC、NBA分别表示石油醚、二氯甲烷、乙酸乙酯、正丁醇萃取层。
(4)流式细胞仪检测细胞周期:取对数生长期的K562细胞,以1.5×105个/mL的细胞密度接种于6孔板中,培养1h后,加入谷精草流分E3,继续培养48h后,收集细胞,用PBS洗2遍后去PBS,用70%乙醇固定,过夜。次日,取出流式样本,离心(2500rpm,5min,4℃),吸弃上清,涡旋震荡使细胞松散。用1×PBS洗涤细胞两次。洗涤后,加入已配好的PI染液,37℃染色30min后,进行细胞周期检测。
结果显示:在谷精草流分E3作用下,K562细胞产生了明显的G2/M期阻滞,并且在50μg/mL作用时出现明显的亚二倍体Sub-G1峰。结果如图2所示。
(5)Annexin-V/PI双染法检测细胞凋亡:收集终浓度为50μg/ml谷精草流分E3处理48h后的细胞,离心(2500rpm,5min,4℃),弃上清。加入1mL冷的PBS轻轻震荡使细胞悬浮,离心,弃上清,重复上述步骤3-4次。将细胞重悬于Binding Buffer 500μL,加入AnnexinV-FITC 5μL以及5μL PI轻轻混匀,避光室温反应15min,FCM检测。
结果显示:随着作用时间的延长,E3能促进Annexin V阳性细胞率明显大幅度增加,说明E3具有很强的诱导K562细胞凋亡的作用。结果图3所示。
(6)谷精草流分E3对极光激酶活性的抑制作用:取指数生长期K562细胞,接种25cm2培养瓶中,取一定浓度谷精草流分E3(12.5、25、50μg/mL)给药,培养箱中培养48h。冰浴中提取细胞组织总蛋白,用考马斯亮蓝法测定蛋白浓度。取30μg样品上样,12%SDS-PAGE凝胶分离,4℃恒流(100mA)电泳转膜2h。取出PNC膜,5%BSA封闭后,与兔抗人Phospho-Aurora A、Phospho-Aurora B、Phospho-AuroraC、Aurora B、Phospho-Histone H3(Ser10)、Histone-H3、β-actin单克隆抗体4℃孵育过夜,山羊抗兔二抗(l:500)室温下孵育2h后,电化学发光免疫分析显色数分钟,暗室曝光,取出底片显影,见附图4所示。
结果显示:谷精草流分E3处理K562细胞24h后,K562细胞中蛋白表达量降低。谷精草流分E3呈剂量依赖性下调Phospho-AuroraA、Phospho-Aurora B及Phospho-Histone H3(Ser10)的磷酸化水平,表明其对极光激酶A/B的活性有很好的抑制作用。
上述结果表明:谷精草酚酸类富集流分E3对K562细胞具有较好的抑制细胞增殖活性,且在测定剂量范围内呈现良好的剂量依赖性关系。对其作用机制进行探讨,发现谷精草酚酸类富集流分E3是通过抑制极光激酶A/B的活性,进而引起细胞周期阻滞、诱导细胞凋亡。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所做的改变、修饰、替代、组合、简化均应为等效的置换方式,都包含在本发明的框架包含范围之内。
Claims (4)
1.一种谷精草酚酸类富集流分,其特征在于:从谷精草中分离出酚酸类富集流分具有活性的化合物如下:
2.一种如权利要求1所述谷精草酚酸类富集流分的制备方法,其特征在于:取谷精草药材10kg,用60%乙醇回流提取3次,每次2小时,回收乙醇,得浸膏;将浸膏分散于水中,以石油醚、二氯甲烷、乙酸乙酯、正丁醇萃取;将乙酸乙酯萃取物经HPD101大孔树脂分离,依次以水E0,10%的乙醇-水E1、30%乙醇-水E2、70%乙醇-水E3以及95%乙醇E4进行洗脱;其中的70%流分为E3;以没食子算为对照品,并采用HPLC法测定了E3中总酚酸的含量17.85%;E3经过反复硅胶色谱、Sephadex LH-20、ODS及制备液相分离手段,并结合理化性质和现代波谱学手段从中分离鉴定出酚酸类单体化合物。
3.一种如权利要求1所述谷精草酚酸类富集流分在制备抗肿瘤药物中的应用。
4.根据权利要求3所述的谷精草酚酸类富集流分在制备抗肿瘤药物中的应用,其特征在于:谷精草酚酸类富集流分对人白血病细胞(K562)的具有抑制增殖活性且作用机制是通过抑制极光激酶A/B的活性,进而引起细胞周期阻滞、诱导细胞凋亡。
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