摘要
目的:分离纯化骆驼刺正丁醇萃取部位中的单体化合物,并探讨其对人宫颈癌HeLa细胞增殖、迁移的影响。方法:采用硅胶柱、Sephadex LH-20凝胶色谱柱、制备型高效液相色谱等方法对骆驼刺正丁醇萃取部位进行分离纯化,根据理化性质和波谱(质谱、氢谱、碳谱等)数据分析、鉴定化合物结构。以人宫颈癌HeLa细胞为对象,以5-氟尿嘧啶(5-FU)为阳性对照,采用四甲基偶氮唑盐法检测经各化合物不同剂量(均为6.25、12.5、25、50、100、200μg/mL)预处理后的细胞抑制率,并计算半数抑制浓度(IC50),以筛选活性单体;采用划痕实验考察上述活性单体(均为50μg/mL)对HeLa细胞迁移能力的影响;采用金氏公式评价5-FU与上述活性单体分别联用[(3.125+6.25)、(6.25+12.5)、(12.5+25)、(25+50)μg/mL]的效果。结果:从骆驼刺正丁醇萃取物部位中共分离得到6个化合物,分别鉴定为紫铆素(Ⅰ)、3′,4′,7-三羟基异黄酮(Ⅱ)、对甲氧基苯乙酸(Ⅲ)、4-羟基苯乙酮(Ⅳ)、橙黄胡椒酰胺(Ⅴ)、原儿茶醛(Ⅵ)。与空白对照组比较,5-FU和各化合物(5-FU:6.25～200μg/mL各剂量,化合物Ⅰ:12.5～200μg/mL各剂量,化合物Ⅱ:25、50、200μg/mL,化合物Ⅲ:6.25、100、200μg/mL,化合物Ⅳ:50、100、200μg/mL,化合物Ⅴ:12.5、25、200μg/mL,化合物Ⅵ:6.25～200μg/mL各剂量)均可显著升高细胞抑制率,且化合物Ⅰ、Ⅴ、Ⅵ的IC50值均显著降低(P<0.05或P<0.01),其中化合物Ⅰ、Ⅵ的IC50值相对较低。5-FU与化合物Ⅰ、Ⅵ组细胞的迁移距离均较空白对照组显著缩小(P<0.05或P<0.01);5-FU分别与化合物Ⅰ、Ⅵ联用后,对HeLa细胞的增殖具有相加或增强的协同抑制作用(增效指数均大于0.9)。结论:化合物Ⅰ～Ⅵ均为首次从骆驼刺属植物中分离得到,紫铆素和原儿茶醛是其正丁醇萃取部位的活性单体。这2种活性单体均可抑制人宫颈癌HeLa细胞的增殖和迁移,具有较强的体外细胞抑制作用,且与5-FU联用后的抑制作用强于两者分别单用。
OBJECTIVE:To separate and purify Alhagi sparsifolia n-butanol extract monomeric compounds,and to investigate its effects on the proliferation and metastasis of human cervical cancer HeLa cells.METHODS:The n-butanol extract was separated and purified by silica gel column,Sephadex LH-20 gel column and prep-HPLC.The structures of compounds were analyzed and identified according to physicochemical properties and spectrum(mass spectrum,hydrogen spectrum,carbon spectrum)data.Using human cervical cancer HeLa cells as objects,5-FU as positive control,MTT assay was used to detect the inhibitory rate of HeLa cells pretreated with different doses of compounds(6.25,12.5,25,50,100,200μg/mL);IC50 was calculated to screen active monomers.Scratch test was used to investigate the effects of above active monomers(all 50μg/mL)on the migration ability of HeLa cells.Kim’s formula was used to evaluate the effects of 5-FU separately combined with above active monomers[(3.125+6.25),(6.25+12.5),(12.5+25),(25+50)μg/mL].RESULTS:Six compounds were isolated from the n-butanol extract part of A.sparsifolia and identified as butin(Ⅰ),3′,4′,7-trihydroxyisoflavone(Ⅱ),p-methoxyphenylacetic acid(Ⅲ),4-hydroxyacetophenone(Ⅳ),aurantiamide acetate(Ⅴ),protocatechualdehydea(Ⅵ).Compared with blank control group,5-FU and each compound(5-FU:6.25-200μg/mL,compoundⅠ:12.5-200μg/mL;compoundⅡ:25,50,200μg/mL;compoundⅢ:6.25,100,200μg/mL;compoundⅣ:50,100,200μg/mL;compoundⅤ:12.5,25,200μg/mL;compoundⅥ:6.25-200μg/mL)could significantly increase the cell inhibition rate.IC50 of compoundⅠ,Ⅴ,Ⅵwere decreased significantly(P<0.05 or P<0.01),and those of compoundⅠandⅥwere lower relatively.The migration distance of cells in 5-FU and compoundⅠandⅥgroups were decreased significantly,compared with blank control group(P<0.05 or P<0.01).5-FU separately combined with compoundⅠandⅥshowed additive and enhanced inhibitory effects on the proliferation of HeLa cells(synergistic index >0.9).CONCLUSIONS:CompoundsⅠ-Ⅵare isolated from Alhagi for the first time.Butin and protocatechualdehydea are active monomers of its n-butanol extract part.Above two monomers can inhibit the proliferation and migration of human cervical cancer Hela cells,with strong inhibitory effect in vitro,and stronger inhibitory effect combined with 5-FU than any compound alone.
作者
刘雪松
马晓玲
石磊岭
阿勒腾图娅
张大鹏
李宁
魏鸿雁
LIU Xuesong;MA Xiaoling;SHI Leiling;Aletengtuya;ZHANG Dapeng;LI Ning;WEI Hongyan(College of Life Sciences and Technology,Xinjiang University,Urumqi 830046,China;Xinjiang Uygur Autonomous Region Institute of Traditional Chinese Medicine and Ethnic Medicine,Urumqi 830002,China;School of Traditional Chinese Medicine,Shenyang Pharmaceutical University,Shenyang 110016,China)
出处
《中国药房》
CAS
北大核心
2019年第4期506-512,共7页
China Pharmacy
基金
国家自然科学基金资助项目(No.81460662)
乌鲁木齐市天山区科学技术计划项目(No.20180012)
关键词
骆驼刺
正丁醇萃取部位
单体化合物
抗肿瘤活性
增殖
迁移
联合用药
人宫颈癌HELA细胞
Alhagi sparsifolia
n-butanol extract part
Monomeric compounds
Antitumor activity
Proliferation
Migration
Drug combination
Human cervical cancer HeLa cells
