CN117384182A - 海洋真菌来源的抗肿瘤活性化合物及其制备方法 - Google Patents
海洋真菌来源的抗肿瘤活性化合物及其制备方法 Download PDFInfo
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Abstract
本发明涉及海洋真菌来源的抗肿瘤活性化合物及其制备方法。所述抗肿瘤活性化合物具有化合物1和2所示的结构:
Description
技术领域
本发明属于真菌活性次级代谢产物领域,具体涉及海洋真菌来源的抗肿瘤活性化合物及其制备方法。
背景技术
海桑果内生真菌(HSG11-9)是从海南红树海桑果(果实)中分离得到,申请人先前自该真菌中分离获得了系列抗肿瘤活性化合物(中国发明专利申请号:202111133295.6),申请人对该真菌发酵物的其他馏分做进一步研究获得两个抗肿瘤活性化合物。
发明内容
本发明提供一种海洋真菌来源的抗肿瘤活性化合物或其药学上可接受的盐,其特征在于所述抗肿瘤活性化合物具有化合物1和2所示的结构:
本发明的另一实施方案提供一种制备化合物1和/或2的方法,其特征在于包括如下步骤:
(1)配制种子培养基,将海桑果内生真菌HSG11-9接入种子培养基,26℃,培养3天得种子培养液;
(2)将步骤(1)得到的种子培养液接入发酵培养基中,26℃恒温静置培养40-45天得发酵物;
(3)将步骤(2)得到的发酵物中的发酵液和菌体分离,发酵液用等体积的乙酸乙酯萃取2~4次,合并萃取液后减压浓缩得到浸膏;经色谱分离得到化合物1和/或2。
步骤(3)中所述色谱分离的步骤为:将浸膏经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0、90:10、80:20、70:30、60:40,50:50、40:60、30:70、20:80、10:90、0:100梯度洗脱,每个梯度收集两个柱体积,合并80:20和60:40梯度洗脱得到的馏分、浓缩后经Sephadex LH-20凝胶柱层析,洗脱剂为氯仿:甲醇=1:1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为CH3OH:H2O=35:65,最终得到化合物1、2。
其中所述洗脱剂或流动相的比例均为体积比;所述种子培养基中含有葡萄糖1.5%–3.0%、酵母膏0.1%–0.5%、蛋白胨0.1%–0.5%、粗海盐0.11%–0.6%、适量的水;所述发酵培养基中含有葡萄糖1.6%–3.5%、酵母膏0.1%–0.5%、蛋白胨0.1%–0.5%、粗海盐0.11%–0.6%、适量的水;上述百分比均为重量百分比;所述种子培养基和发酵培养基均需120℃灭25–30分钟。
本发明的另一实施方案提供一种利用上述海桑果内生真菌HSG11-9在制备抗肿瘤化合物1和/或2中的应用。优选针对Hela肿瘤细胞。
本发明所述海桑果内生真菌(HSG11-9)的菌种保藏信息:保藏单位名称:中国微生物菌种保藏管理委员会普通微生物中心;保藏单位地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所;保藏日期:2021年9月8日;保藏编号:CGMCC No.23226;分类命名:土曲霉Aspergillus terreus。(中国发明专利申请号:202111133295.6)
本发明的另一实施方案提供上述化合物1、2或其药学上可接受的盐在制备抗肿瘤药物中的应用。
本发明提供一种抗肿瘤药物组合物,其特征在于以上述化合物1、2或其药学上可接受的盐作为有效成分。
本发明提供的上述抗肿瘤药物组合物,还可包含其他抗肿瘤药物;也可以包含药学上可接受的辅料(优选药学上可接受的载体、稀释剂或赋形剂)。上述药物组合物的剂型可以是固体制剂、半固体制剂或液体制剂。
本发明中术语“药学上可接受的盐”是指非毒性的无机或有机酸和/或碱的加成盐,可参见“Salt selection for basic drugs”,Int.J.Pharm.(1986),33,201–217。
附图说明
图1是化合物1的COSY、HMBC相关图;
图2是化合物1的1H NMR图;
图3是化合物1的13C NMR图;
图4是化合物1的DEPT图;
图5是化合物1的H-H COSY图;
图6是化合物1的HSQC图;
图7是化合物1的HMBC图;
图8是化合物1的NOESY图。
具体实施方式
为了便于对本发明的进一步理解,下面提供的实施例对其做了更详细的说明。但是这些实施例仅供更好的理解发明而并非用来限定本发明的范围或实施原则,本发明的实施方式不限于以下内容。
实施例1
(1)海桑果内生真菌HSG11-9的菌种培养
配制种子培养基:葡萄糖20g,蛋白胨2g,酵母膏2g,粗海盐2.5g,水1.0L,平均分装于2个1000mL锥形瓶,120℃灭25分钟。
将海桑果内生真菌HSG11-9菌株接入配制好的种子培养基中,于26℃下恒温培养3天得种子培养液;
(2)海桑果内生真菌HSG11-9的发酵
配制发酵培养基:葡萄糖1.1kg,蛋白胨100g,酵母膏100g,海盐125g,水50L,平均分装于100个1000mL锥形瓶中,120℃灭25–30分钟。
取适量的步骤(1)中得到的种子培养液(10mL/瓶)接入装有发酵培养基的锥形瓶中,于26℃恒温静置培养45天得发酵物。
(3)浸膏的制备
将步骤(2)得到的发酵物中的发酵液和菌体分离,发酵液用等体积的乙酸乙酯萃取3次,合并萃取液后减压浓缩得到浸膏(26.3g);
(4)化合物1、2的提取分离
步骤(3)得到的浸膏经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0、90:10、80:20、70:30、60:40,50:50、40:60、30:70、20:80、10:90、0:100梯度洗脱,每个梯度收集两个柱体积,合并80:20和60:40梯度洗脱得到的馏分、浓缩后经Sephadex LH-20凝胶柱层析,洗脱剂为氯仿:甲醇=1:1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为CH3OH:H2O=35:65,最终得到化合物1(18.3mg)、2(15.2mg)。
化合物1:白色粉末,254nm紫外灯照射有吸收,高分辨质谱HR-ESI-MS(m/z347.2501,[M+H]+,计算为C22H35O3 +,347.2581)提示分子式为C22H34O3,不饱和度为6。1H NMR谱(表)显示该化合物有6个亚甲基(δH 2.82,1H;2.32,1H;2.10,2H;2.05,2H;1.95,2H;1.42,4H;),6个次甲基,5个甲基(δH 1.37,3H;0.88,3H)。13C NMR和DEPT谱显示该化合物共有22个碳信号,其中有5个季碳(δC 139.45,135.39,134.26,71.28,64.33),6个亚甲基碳(δC43.53,40.06,39.72,31.15,26.05,22.67),5个甲基碳(δC 29.34,29.26,19.12,16.40,16.16)。经文献查阅,化合物1的核磁数据与化合物yanuthone M的核磁数据基本一致,其主要区别为C-18的羰基被环氧基取代。故化合物1的结构鉴定为2,4,6-trimethyl-12-(14,15,13,18-diepoxy-16-methyl-16-cyclohexenyl)-6,10-dodecadie n-2-ol。
化合物2:白色固体。1H NMR(CD3OD,400MHz):7.27(1H,ddm,J=15.2,10.1Hz,H-9),6.73(1H,d,J=15.0Hz,H-8),4.14(1H,d,J=7.0Hz,H-4),6.31(2H,m,H-11),3.80(1H,dd,J=11.0,2.5Hz,H-6),3.55(1H,dd,J=11.0,5.5Hz,H-6),3.51(1H,ddd,J=7,5.5,2.5Hz,H-5),6.25(1H,m,H-10),2.21(2H,m,H-12),1.29(12H,m,H-14,15,16,17,18,19),1.45(2H,q,J=7.5Hz,H-13),0.89(3H,t,J=7Hz,H-20),.13C NMR(CD3OD,100MHz):δC 198.74(C-7),175.31(C-2),145.50(C-9),124.22(C-8),88.08(C-3),79.05(C-4),148.62(C-11),62.09(C-6),60.34(C-5),130.49(C-10),34.22(C-12),33.04(C-18),30.66,30.55,30.43,30.31(C-14,15,16,17),29.84(C-13),23.72(C-19),14.43(C-20)。经文献查阅,化合物2的核磁数据与已知化合物Pramanicin A的核磁数据基本一致,因此化合物2的结构鉴定为Pramanicin A。
实施例2
(1)海桑果内生真菌HSG11-9的菌种培养
配制种子培养基(10.0L):葡萄糖1.5%(重量百分比,下同)、酵母膏0.5%、蛋白胨0.1%、粗海盐0.11%,其余为水;平均分装于16个1000mL锥形瓶,120℃灭25–30分钟。
将海桑果内生真菌HSG11-9接入配制好的种子培养基中,于26℃恒温培养3天得种子培养液;
(2)海桑果内生真菌HSG11-9的发酵
配制发酵培养基(100L):葡萄糖1.6%(重量百分比,下同)、酵母膏0.5%、蛋白胨0.1%、粗海盐0.11%,其余为水;平均分装于200个1000mL锥形瓶,120℃灭30分钟。
取适量的步骤(1)中得到的种子培养液接入装有发酵培养基的锥形瓶中,于26℃静置培养40天得发酵物。
(3)浸膏的制备
将步骤(2)得到的发酵物中的发酵液和菌体分离,发酵液用等体积的乙酸乙酯萃取4次,合并萃取液后减压浓缩得到浸膏;
(4)化合物1、2的提取分离
步骤(3)得到的浸膏经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0、90:10、80:20、70:30、60:40,50:50、40:60、30:70、20:80、10:90、0:100梯度洗脱,每个梯度收集两个柱体积,合并80:20和60:40梯度洗脱得到的馏分、浓缩后经Sephadex LH-20凝胶柱层析,洗脱剂为氯仿:甲醇=1:1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为CH3OH:H2O=35:65,最终得到化合物1、2。
实施例3
(1)海桑果内生真菌HSG11-9的菌种培养
配制种子培养基(1.0L):葡萄糖3.0%(重量百分比,下同)、酵母膏0.1%、蛋白胨0.5%、粗海盐0.6%,其余为水;平均分装于3个500mL锥形瓶,120℃灭25–30分钟。
将海桑果内生真菌HSG11-9菌株接入配制好的种子培养基中,于28℃恒温培养3天得种子培养液;
(2)海桑果内生真菌HSG11-9的发酵
配制发酵培养基(10L):葡萄糖3.5%(重量百分比,下同)、酵母膏0.1%、蛋白胨0.5%、粗海盐0.6%,其余为水;平均分装于20个1000mL锥形瓶,120℃灭25分钟。
取适量的步骤(1)中得到的种子培养液接入装有发酵培养基的锥形瓶中,于26℃恒温静置培养42天得发酵物。
(3)浸膏的制备
将步骤(2)得到的发酵物中的发酵液和菌体分离,发酵液用等体积的乙酸乙酯萃取2次,合并萃取液后减压浓缩得到浸膏;
(4)化合物1、2的提取分离
步骤(3)得到的浸膏经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0、90:10、80:20、70:30、60:40,50:50、40:60、30:70、20:80、10:90、0:100梯度洗脱,每个梯度收集两个柱体积,合并80:20和60:40梯度洗脱得到的馏分、浓缩后经Sephadex LH-20凝胶柱层析,洗脱剂为氯仿:甲醇=1:1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为CH3OH:H2O=35:65,最终得到化合物1、2。
实施例4细胞毒活性测试
使用MTT法,测试了化合物1对肿瘤细胞株Hela细胞毒活性。取处于指数生长期的肿瘤细胞,加入0.02%Trypsin-EDTA使贴壁细胞脱落,用含10%胎牛血清的RPMI1640培养液配成单细胞悬液,计数并调整细胞数后接种于96孔板,置于37℃二氧化碳培养箱内培养24h。将待测化合物分设成2.00,5.00,10.00,20.00μg/mL,每组3个平行。待测样品用DMSO助溶,RPMI1640稀释后加入96孔板,37℃二氧化碳培养箱内培养72h。MTT用无血清RPMI1640溶解,每孔加50μL。置于37℃二氧化碳培养箱内培养4h,取出,吸出上清液,每孔加入DMSO150μL溶解生成的甲臜,用酶标仪630nm处测定吸光值并计算相应抑制百分率和IC50值。
化合物1对肿瘤细胞Hela体外抑制作用(n=3)
Claims (9)
1.一种海洋真菌来源的抗肿瘤活性化合物或其药学上可接受的盐,其特征在于所述抗肿瘤活性化合物具有化合物1和2所示的结构:
2.一种制备化合物1和/或2的方法,其特征在于包括如下步骤:
(1)配制种子培养基,将海桑果内生真菌HSG11-9接入种子培养基,26℃,培养3天得种子培养液;
(2)将步骤(1)得到的种子培养液接入发酵培养基中,26℃恒温静置培养40-45天得发酵物;
(3)将步骤(2)得到的发酵物中的发酵液和菌体分离,发酵液用等体积的乙酸乙酯萃取2~4次,合并萃取液后减压浓缩得到浸膏;经色谱分离得到化合物1和/或2。
3.权利要求2所述的方法,其特征在于步骤(3)中所述色谱分离的步骤为:将浸膏经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0、90:10、80:20、70:30、60:40,50:50、40:60、30:70、20:80、10:90、0:100梯度洗脱,每个梯度收集两个柱体积,合并80:20和60:40梯度洗脱得到的馏分、浓缩后经Sephadex LH-20凝胶柱层析,洗脱剂为氯仿:甲醇=1:1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为CH3OH:H2O=35:65,最终得到化合物1、2。
4.海桑果内生真菌HSG11-9在制备抗肿瘤化合物1和/或2中的应用。优选针对Hela肿瘤细胞。
5.权利要求1所述的化合物1、2或其药学上可接受的盐在制备抗肿瘤药物中的应用。
6.一种抗肿瘤药物组合物,其特征在于以上述化合物1、2或其药学上可接受的盐作为有效成分。
7.权利要求6所述的药物组合物,其特征在于还可包含其他抗肿瘤药物。
8.权利要求6-7任一项所述的药物组合物,其特征在于也可以包含药学上可接受的辅料(优选药学上可接受的载体、稀释剂或赋形剂)。
9.权利要求6-8任一项所述的药物组合物,其特征在于上述药物组合物的剂型可以是固体制剂、半固体制剂或液体制剂。
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