CN113527325A - 一种尼日利亚菌素衍生物及其制备方法和在制备抗肿瘤药物中的应用 - Google Patents
一种尼日利亚菌素衍生物及其制备方法和在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
Description
技术领域:
本发明属于天然产物领域,具体涉及一种尼日利亚菌素衍生物及其制备方法和在制备抗肿瘤药物中的应用。
背景技术:
据世界卫生组织国际癌症研究机构(IARC)公布数据显示,2020年全球新发癌症病例1929万例,癌症死亡病例996万例,其中我国新发病例和死亡人数均位居全球第一。2020年全球男性新发癌症1007万例,占总人数的52%,其中前列腺癌(Prostate Cancer,PCa)是一种威胁全球男性健康的重大疾病。2020年最新数据显示,男性新发癌症中前列腺癌患癌人数达141万,仅次于第一位的肺癌(144万)。多年来,研究者致力于寻取新型、高效、低毒的抗肿瘤化合物,天然产物是研发新型抗肿瘤药物等人类重大疾病用药的宝贵资源,特别是海洋来源天然产物,从中筛选发现药物候选物,其活性要远高于许多其它成药来源的化合物,在抗肿瘤药物研发过程中具有举止轻重的地位。我国已上市的海洋类药物治疗领域主要集中于心血管类药物,抗肿瘤用途的海洋类药物亟需开发。因此,本发明人从海洋微生物中筛选发现具有良好抗肿瘤活性的天然产物对于抗前列腺癌药物研发具有重要意义。
发明内容:
本发明的第一个目的是提供一种化合物,所述化合物为29-O-methylnigericin,其结构如式(Ⅰ):
本发明人通过对一株海洋链霉菌HNM0561摇床放大发酵和发酵提取物提取纯化,从中得到化合物1。经结构分析,化合物1被确定为尼日利亚菌素29-O-methylnigericin,为本发明人首筛选并分离纯化的天然产物。通过对化合物1的抗肿瘤活性评价,发现化合物1对五种人前列腺癌细胞PC3、22RV1、LNCaP、DU145和VCaP显示出较好的抑制作用,尤其是前列腺癌细胞PC3、22RV1和LNCaP的IC50分别达到了1.69,1.91和0.73μM,可以做为抗前列腺癌药物开发的候选先导化合物。
本发明的第二个目的是提供所述化合物29-O-methylnigericin或其药用盐在制备抗肿瘤药物中的应用。优选地,所述肿瘤为前列腺癌。
本发明的第三个目的是提供一种化合物29-O-methylnigericin的制备方法,包括以下步骤:
1)将海洋链霉菌Streptomyces sp.HNM0561接种到发酵培养基中培养,获得发酵培养物;
2)分离发酵液和菌丝体,将发酵液用乙酸乙酯萃取,乙酸乙酯萃取液浓缩得发酵液浸膏;
3)菌丝体经丙酮水溶液浸提,提取液经浓缩后,再用乙酸乙酯萃取,乙酸乙酯萃取液浓缩得菌丝体浸膏;
4)合并发酵液浸膏和菌丝体浸膏,用反相硅胶分离,采用甲醇:水从10:90,20:80,30:70,50:50,70:30,80:20,90:10,100:0,v/v梯度洗脱顺序得8个组分Fr1-Fr8,收集甲醇:水80:20v/v洗脱的组分Fr6,经Sephadex LH-20柱层析,以甲醇为洗脱剂,洗脱纯化后的产物,经高效液相纯化得到尼日利亚菌素29位甲基化衍生物29-O-methylnigericin。
在一优选例中,所述的制备海洋链霉菌Streptomyces sp.HNM0561的发酵培养物是将海洋链霉菌Streptomyces sp.HNM0561接种到发酵培养基中培养,所述的发酵培养基每1000mL含有:葡萄糖20g,酵母膏10g,牛肉膏3g,玉米浆3g,可溶性淀粉10g,磷酸氢二钾0.5g,硫酸镁0.5g,碳酸钙2g,余量为水。
另一优选例中,所述的经高效液相纯化是以210nm和255nm波长做检测、采用4ml/min的流速,以甲醇:水=75:25,v/v进行等梯度洗脱进行半制备高效液相分离,HPLC柱子为YMC-pack ODS-A,10×250mm,5μm,于保留时间tR 29.1min得到尼日利亚菌素29位甲基化衍生物29-O-methylnigericin。
本发明的第四个目的是提供一种药物,包括有效量的作为活性成分的所述化合物29-O-methylnigericin或其药用盐。
本发明的第五个目的是提供一种海洋链霉菌Streptomyces sp.HNM0561,其保藏编号为CCTCC NO:M 2020255。
本发明的第六个目的是提供海洋链霉菌Streptomyces sp.HNM0561在制备上述化合物29-O-methylnigericin中的应用。
与现有技术相比,本发明具有如下有益效果:
(1)本发明筛选并分离纯化出具有抗肿瘤活性的天然物质尼日利亚菌素29位甲基化衍生物29-O-methylnigericin;
(2)本发明29-O-methylnigerici能够明显抑制肿瘤细胞增殖活性和迁移,诱导肿瘤细胞凋亡;且抗肿瘤作用比一些现有药物更强。
本发明的Streptomyces sp.HNM0561于2020年06月30日保藏于中国典型培养物保藏中心(CCTCC),地址:中国.武汉.武汉大学,邮编:430072,保藏编号:CCTCC NO:M2020255。
附图说明:
图1是29-O-methylnigericin(1)主要的1H-1H COSY,和HMBC信息;
图2是29-O-methylnigericin对PCa细胞克隆形成的影响;
图3是29-O-methylnigericin对22Rv1细胞凋亡的影响;
图4是29-O-methylnigericin对PC-3细胞凋亡的影响;
图5是29-O-methylnigericin对PC-3细胞迁移的影响。
具体实施方式:
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1:化合物1的制备
1.微生物培养条件:
配置培养基:取葡萄糖20g,酵母膏10g,牛肉膏3g,玉米浆3g,可溶性淀粉10g,磷酸氢二钾0.5g,硫酸镁0.5g,碳酸钙2g,然后溶于适量的水中,用水定容至1000mL,121℃高温灭菌20min,备用。
将海洋链霉菌HNM0561接种到上述培养基中,28℃条件下摇床培养3天,得种子培养液,再将种子培养液按照1%的体积比接种到上述培养基中,28℃条件下摇床培养11天,得到海洋链霉菌HNM0561的发酵产物。
2.提取分离:
将上述海洋链霉菌HNM0561的发酵产物,以3600rpm离心,得上清发酵液和沉淀菌丝体。上清发酵液用乙酸乙酯等体积萃取3次,乙酸乙酯萃取液在低于40℃减压浓缩得发酵液浸膏;沉淀菌丝体用体积分数85%的丙酮水溶液超声浸提,提取液经减压蒸馏后,再用乙酸乙酯反复萃取三次,乙酸乙酯萃取液在低于40℃下减压浓缩得菌丝体浸膏;合并发酵液浸膏和菌丝体浸膏共计得约27.6g浸膏。该浸膏用反相硅胶分离,经拌样、干法装柱后,采用甲醇:水(10:90,20:80,30:70,50:50,70:30,80:20,90:10,100:0,v/v)梯度洗脱顺序得8个组分(Fr1-Fr8)。组分Fr6(甲醇:水80:20v/v洗脱的组份)经Sephadex LH-20柱层析,以甲醇为洗脱剂,洗脱纯化后的产物,以210nm和255nm波长做检测、采用4mL/min的流速,以甲醇:水(75:25,v/v)进行等梯度洗脱进行半制备高效液相(YMC-pack ODS-A,10×250mm,5μm)分离,得到化合物1(4.0mg,保留时间tR 29.1min)。
实施例2化合物1的结构鉴定
对化合物1进行高分辨质谱(HRESIMS)、一维和二维核磁共振(NMR)等数据测试,从而确定化合物1的化学结构。
化合物1呈无色油状;高分辨质谱HRESIMS m/z 761.4822[M+Na]+(calcd forC41H70NaO11),建议分子式为C41H70O11,不饱和度为7;1H和13C NMR数据见表1;核磁数据显示分子中含有41个碳原子,其中包括9个甲基,2个甲氧基,10个亚甲基,15个次甲基,5个季碳。观察一维和二维氢谱发现,分子中无芳香碳或者烯烃碳,且碳谱中出现多个连氧次甲基或者连氧饱和积碳,推测该化合物是一个聚醚类化合物。1H-1H COSY谱图中显示含有:
H3-31/H-2/H-3/H-4(H3-32)/H2-5/H2-6/H-7/H2-8/H-9/H2-10/H-12/H3-33;
H3-34/H-14/H2-15;
H-17/H2-18/H2-19;
H-21/H-22(H3-37)/H2-23/H-24/H-25/H-26(H3-38)/H2-27/H-28/H3-39四段自旋耦合体系。结合HMBC碳氢远程相关,发现化合物1与尼日利亚菌素nigericin非常相似。仔细比较差异发现化合物1在尼日利亚菌素29位羟基变成了羟甲基,同时H3-41到C-29的HMBC信号可以证明上面的推断。经SciFinder检索,化合物1为结构崭新的化合物,命名为29-O-methylnigericin(OMN)。化合物1主要的1H-1H COSY和HMBC信息见图1。
表1. 29-O-methylnigericin的核磁共振谱数据(700MHz,DMSO-d6,ppm)
化合物1的结构式如式(I)所示,命名为29-O-methylnigericin(OMN):
实施例3 29-O-methylnigericin具有抑制前列腺癌细胞活性作用
选用对数生长期的LNCaP、22Rv1、PC-3、DU145、VCaP细胞,用胰蛋白酶消化,调整细胞浓度为5.0×104个/mL后接种到96孔板,每孔100μL,然后放置在37℃,5%CO2培养箱中培养。待细胞完全贴壁后,加入OMN(设置6个浓度梯度10-5M、10-6M、10-7M、10-8M、10-9M、10-10M,每个浓度设置6个复孔,重复实验3次),空白对照组加入含1‰DMSO的完全培养液(LNCaP、22Rv1、VCaP细胞用10%FBS RPMI 1640,PC-3细胞用10%FBS DMEM F12,DU145细胞用10%FBS MEM)。于化合物作用后的24、48、72小时,加入MTT试剂(索莱宝,M8180-250mg),检测各孔OD570值,按(药物处理孔OD值-空白孔OD值)/(对照组OD值-空白孔OD值)公式计算细胞存活率,然后使用GraphPad Prism 6软件拟合IC50。结果如表2所示,说明29-O-methylnigericin对前列腺癌细胞的活性具有明显抑制作用。
表2.OMN对PCa细胞活性的影响
实施例4 29-O-methylnigericin具有抑制前列腺癌细胞增殖作用
选用对数生长期的22Rv1、PC-3细胞,用胰蛋白酶消化,调整细胞浓度为1.0×103个/mL,将细胞接种到6孔板,每孔1mL细胞悬液,再加1mL完全培养液(22Rv1细胞用10%FBSRPMI 1640,PC-3细胞用10%FBS DMEM F12)。然后放置在37℃,5%CO2培养箱中培养。待细胞完全贴壁后,加入OMN(设置3个浓度梯度0.1μM、1μM、10μM,每个浓度设置3个复孔),阳性对照组1加入1μM多西他赛(docetaxel)、阳性对照组2加入1μM比卡鲁胺(bicalutamide),空白对照组加入含1‰DMSO的完全培养液(22Rv1细胞用10%FBS RPMI 1640,PC-3细胞用10%FBS DMEM F12)。培养2周左右细胞形成明显克隆,终止培养,用结晶紫染色,再用菌落计数仪拍照观察平板克隆形成情况。
检测结果如图2所示,A所示,0.1μM,1μM,10μM 29-O-methylnigericin均可以显著抑制22Rv1细胞平板克隆形成,说明29-O-methylnigericin具有很好的抑制22Rv1细胞增殖作用;B所示,0.1μM,1μM,10μM 29-O-methylnigericin均可以显著抑制PC-3细胞平板克隆形成,说明29-O-methylnigericin具有很好的抑制PC-3细胞增殖作用。
实施例5 29-O-methylnigericin具有诱导前列腺癌细胞凋亡作用
选用对数生长期的22Rv1、PC-3细胞,用胰蛋白酶消化,调整细胞浓度为5.0×105个/mL,将细胞接种到6孔板,每孔1mL细胞悬液,再加1mL完全培养液(22Rv1细胞用10%FBSRPMI 1640,PC-3细胞用10%FBS DMEM F12)。然后放置在37℃,5%CO2培养箱中培养。待细胞完全贴壁后,加入OMN(设置3个浓度梯度0.1μM、1μM、10μM,每个浓度设置3个复孔,重复实验3次),空白对照组加入含1‰DMSO的完全培养液(22Rv1细胞用10%FBS RPMI 1640,PC-3细胞用10%FBS DMEM F12),阳性对照组1μM多西他赛。于化合物作用后的48小时按eBioscienceTM Annexin V-FITC Apoptosis Detection Kit(Invitrogen,BMS500FI-300)说明书收集细胞、制备样品,用流式细胞仪检测细胞凋亡情况。
22Rv1、PC-3检测结果分别如图3、图4所示,0.1μM,1μM,10μM 29-O-methylnigericin均以剂量依赖的方式诱导22Rv1、PC-3凋亡,说明29-O-methylnigericin具有很好的诱导前列腺癌细胞凋亡作用。
实施例6 29-O-methylnigericin具有抑制前列腺癌细胞迁移作用
按照xCELLigence RTCA DP Instrument细胞迁移实验方案,准备好CIM-Plate 16检测板,在下室加入165μL含不同浓度化合物的培养液(10%FBS DMEM F12),然后选用对数生长期的PC-3细胞,用胰蛋白酶消化,调整细胞浓度为25.0×105个/mL,将细胞接种到CIM-Plate 16孔板上室,每孔0.1mL细胞悬液,再加14.4μL 10培养液。然后将CIM-Plate16检测板安装到xCELLigence RTCA DP Instrument,放置于37℃,5%CO2培养箱中培养72小时,培养期间用RTCA Software Pro软件对细胞的迁移进行连续72小时监测。
检测结果如图5所示,0.3125μM,0.625μM,1.25μM,2.5μM,5μM,10μM29-O-methylnigericin以剂量依赖的方式抑制PC-3细胞迁移,通过拟合得到抑制迁移IC50为0.76μM。
因此,基于尼日利亚菌素29位甲基化衍生物29-O-methylnigericin对五种人前列腺癌细胞具有良好的抑制活性,其可以作为抗前列腺癌药物开发的先导化合物。
Claims (10)
2.权利要求1所述的化合物29-O-methylnigericin或其药用盐在制备抗肿瘤药物中的应用。
3.如权利要求2所述的应用,其特征在于,所述肿瘤为前列腺癌。
4.一种权利要求1所述化合物29-O-methylnigericin的制备方法,其特征在于,包括以下步骤:
1)将海洋链霉菌Streptomyces sp.HNM0561接种到发酵培养基中培养,获得发酵培养物;
2)分离发酵液和菌丝体,将发酵液用乙酸乙酯萃取,乙酸乙酯萃取液浓缩得发酵液浸膏;
3)菌丝体经丙酮水溶液浸提,提取液经浓缩后,再用乙酸乙酯萃取,乙酸乙酯萃取液浓缩得菌丝体浸膏;
4)合并发酵液浸膏和菌丝体浸膏,用反相硅胶分离,采用甲醇:水从10:90,20:80,30:70,50:50,70:30,80:20,90:10,100:0,v/v梯度洗脱顺序得8个组分Fr1-Fr8,收集甲醇:水80:20v/v洗脱的组分Fr6,经Sephadex LH-20柱层析,以甲醇为洗脱剂,洗脱纯化后的产物,经高效液相纯化得到尼日利亚菌素29位甲基化衍生物29-O-methylnigericin。
5.根据权利要求4所述的制备方法,其特征在于,所述的发酵培养基每1000mL含有:葡萄糖20g,酵母膏10g,牛肉膏3g,玉米浆3g,可溶性淀粉10g,磷酸氢二钾0.5g,硫酸镁0.5g,碳酸钙2g,余量为水。
6.根据权利要求4所述的制备方法,其特征在于,所述的经高效液相纯化是以210nm和255nm波长做检测、采用4ml/min的流速,以甲醇:水=75:25,v/v进行等梯度洗脱进行半制备高效液相分离,柱子为YMC-pack ODS-A,10×250mm,5μm,于保留时间tR 29.1min得到尼日利亚菌素29位甲基化衍生物29-O-methylnigericin。
7.一种药物,其特征在于,包含有效量的作为活性成分的权利要求1中所述的化合物29-O-methylnigericin或其药用盐。
8.根据权利要求7所述的药物,其特征在于,所述的药物是抗前列腺癌的药物。
9.海洋链霉菌Streptomyces sp.HNM0561,保藏编号为CCTCC NO:M 2020255。
10.权利要求9所述的海洋链霉菌Streptomyces sp.HNM0561在制备权利要求1所述的化合物29-O-methylnigericin中的应用。
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