骆驼刺正丁醇萃取部位中单体化合物的分离纯化及其对人宫颈癌HeLa细胞的影响研究
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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 investigateits effects on the proliferation and metastasis of human cervical cancer HeLa cells. METHODS:The n-butanol extract was separatedand 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 (6.25,12.5,25,50,100,200 μg/mL);IC50 was calculated to screen active monomers. Scratch test was used to investigate the effects of above active monomers (all 50 μg/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:6.25-200 μg/mL,compound Ⅰ:12.5-200 μg/mL;compound Ⅱ:25,50,200 μg/mL;compound Ⅲ:6.25,100,200 μg/mL;compound Ⅳ :50,100,200 μg/mL;compound Ⅴ :12.5,25,200 μg/mL;compound Ⅵ :6.25-200 μg/mL) could significantly increase the cell inhibition rate. IC50 of compound Ⅰ,Ⅴ,Ⅵ were decreased significantly (P<0.05 or P<0.01),and those of compound Ⅰ and Ⅵ were lower relatively. The migration distance of cells in 5-FU and compound Ⅰ and Ⅵgroups were decreased significantly,compared with blank control group (P<0.05 or P<0.01). 5-FU separately combined with compound Ⅰand Ⅵ showed additive and enhanced inhibitory effects on the proliferation of HeLa cells (synergistic index>0.9).CONCLUSIONS:Compounds Ⅰ-Ⅵ are 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.
OBJECTIVE:To separate and purify Alhagi sparsifolia n-butanol extract monomeric compounds,and to investigateits effects on the proliferation and metastasis of human cervical cancer HeLa cells. METHODS:The n-butanol extract was separatedand 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 (6.25,12.5,25,50,100,200 μg/mL);IC50 was calculated to screen active monomers. Scratch test was used to investigate the effects of above active monomers (all 50 μg/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:6.25-200 μg/mL,compound Ⅰ:12.5-200 μg/mL;compound Ⅱ:25,50,200 μg/mL;compound Ⅲ:6.25,100,200 μg/mL;compound Ⅳ :50,100,200 μg/mL;compound Ⅴ :12.5,25,200 μg/mL;compound Ⅵ :6.25-200 μg/mL) could significantly increase the cell inhibition rate. IC50 of compound Ⅰ,Ⅴ,Ⅵ were decreased significantly (P<0.05 or P<0.01),and those of compound Ⅰ and Ⅵ were lower relatively. The migration distance of cells in 5-FU and compound Ⅰ and Ⅵgroups were decreased significantly,compared with blank control group (P<0.05 or P<0.01). 5-FU separately combined with compound Ⅰand Ⅵ showed additive and enhanced inhibitory effects on the proliferation of HeLa cells (synergistic index>0.9).CONCLUSIONS:Compounds Ⅰ-Ⅵ are 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.
引文
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[17]QIONG MX,ZHAN S,WEN JH,et al.Antitumor activity of Pulsatilla chimensis(Bunge)Regel saponins in human liver tumor 7402 cells in vitro and in vivo[J].Phytomedicine,2012,19(3/4):293-300.
[18]梁迎春.白头翁皂苷单体抗肿瘤生长、转移作用及其机制研究[D].北京:北京中医药大学,2016.
[19]邓丽芬,王艳红,贾庆安,等.索拉非尼联合5-氟尿嘧啶对人肝癌细胞株MHCCLM3增殖的抑制作用及其机制[J].中华肝脏病杂志,2013,21(11):845-849.
[20]常明向,吴梅梅,李瀚旻.姜黄素与甘草次酸联用对肝癌HepG2细胞增殖的抑制作用[J].药物评价研究,2017,40(1):42-47.
[21]赵萍萍,霍仕霞,彭晓明,等.驱虫斑鸠菊中紫铆素、紫铆花素的分离及其对人A375黑素瘤细胞黑素合成作用的影响[J].中国药学杂志,2013,48(20):1724-1727.
[22]赵焕新,白虹,李巍,等.苏木化学成分的研究[J].食品与药品,2010,12(5):176-180.
[23]邵红军,房立真,刘吉开.西澳粘滑菇的化学成分研究[J].天然产物研究与开发,2010,22(5):786-788.
[24]石磊岭,马国需,杨峻山,等.天山假狼毒的化学成分研究[J].中草药,2016,47(2):223-226.
[25]汤俊,SUPINYA T,王峥涛,等.蚬壳花椒茎中的橙黄胡椒酰胺[J].中国药学,2003,12(4):231-233.
[26]刘世杰.毛支清口服液的质量控制、制备工艺及抗炎作用研究[D].北京:中央民族大学,2013.
[27]张怀念,张咏莉.宫颈癌分子遗传机制及其相关基因功能研究[J].安徽医药,2015,19(2):219-221.
[28]李津.氨肽酶N抑制剂与5-FU及其衍生物联合抗肿瘤活性研究[D].济南:山东大学,2015.
[29]MUHAMMAD K.PD-L1单链抗体的筛选、抗体偶联药物的制备以及抗肿瘤活性的研究[D].杭州:浙江大学,2018.
[30]李希哲,黄海燕,赵威,等.桂枝化合物的分离与鉴定及神经保护作用[J].沈阳药科大学学报,2016,33(1):14-19.
[31]谢雁鸣,王连心,王永炎.临床联合用药机制研究的探讨[J].中国中药杂志,2014,39(18):3424-3426.
[2]韩曾娇.刺糖多糖对巨噬细胞RAW264.7的免疫调节及信号通路的研究[D].乌鲁木齐:新疆医科大学,2017.
[3]波拉提·马卡比力,熊元君,贾晓光,等.骆驼刺糖中黄酮、异黄酮类化学成分研究[J].新疆中医药,2010,28(2):53-55.
[4]刘静,余梦红,张丛敏,等.紫杉醇联合洛铂经介入栓塞给药化疗宫颈癌的临床观察[J].中国药房,2017,28(17):2374-2377.
[5]LERTKHACHONSUK AA,YIP CH,KHUHAPREMA T,et al.Cancer prevention in Asia:resource-stratified guidelines from the Asian Oncology Summit 2013[J].Lancet Oncol,2013,14(12):e497-e507.
[6]MOHANTY G,GHOSH SK.Risk factors for cancer of cervix,status of screening and methods for its detection[J].Arch Gynecol Obstet,2015,291(2):247-249.
[7]孙婧,金卢阳,王慧莹,等.Eps8与恶性肿瘤增殖、转移和预后的相关研究进展[J].中国实验血液学杂志,2013,21(2):493-497.
[8]张贵杰,李宁,熊元君,等.骆驼刺的化学成分研究[J].中国药学杂志,2009,44(12):897-899.
[9]马晓玲,魏鸿雁,徐晓琴,等.骆驼刺提取物体内外抗肿瘤作用及机制研究[J].天然产物研究与开发,2015,27(3):517-520.
[10]马晓玲,魏鸿雁,贾晓光,等.骆驼刺胶囊对大鼠长期毒性的实验研究[J].中药药理与临床,2014,30(2):113-115.
[11]马晓玲,徐建国,夏提古丽·阿不利孜,等.骆驼刺提取物对免疫抑制小鼠迟发型超敏反应的作用[J].新疆医学,2017,47(10):1103-1105.
[12]FAN L,STRASSER-WEIPPL K,LI JJ,et al.Breast cancer in China[J].Lancet Oncol,2014,15(7):e279-e289.
[13]WU C,QIU S,LIU P,et al.Rhizoma amorphophalli inhibits TNBC cell proliferation,migration,invasion and metastasis through the PI3K/Akt/mTOR pathway[J].J Ethnopharmacol,2018.DOI:10.1016/j.jep.2017.09.033.
[14]HE K,HE J,WANG S,et al.HSHIP induces S-phase arrest and growth inhibition in cervical cancer HeLa cells[J].J Genet Genomics,2010,37(4):249-255.
[15]徐叔云,卞如濂,陈修.药理实验方法学[M].3版.北京:人民卫生出版社,2005:202-204.
[16]刘庭波,李秀琴,王文峰,等.大黄素衍生物E11的筛选及对多发性骨髓瘤细胞增殖抑制和诱导凋亡作用的研究[J].中国实验血液学杂志,2018,26(5):1407-1413.
[17]QIONG MX,ZHAN S,WEN JH,et al.Antitumor activity of Pulsatilla chimensis(Bunge)Regel saponins in human liver tumor 7402 cells in vitro and in vivo[J].Phytomedicine,2012,19(3/4):293-300.
[18]梁迎春.白头翁皂苷单体抗肿瘤生长、转移作用及其机制研究[D].北京:北京中医药大学,2016.
[19]邓丽芬,王艳红,贾庆安,等.索拉非尼联合5-氟尿嘧啶对人肝癌细胞株MHCCLM3增殖的抑制作用及其机制[J].中华肝脏病杂志,2013,21(11):845-849.
[20]常明向,吴梅梅,李瀚旻.姜黄素与甘草次酸联用对肝癌HepG2细胞增殖的抑制作用[J].药物评价研究,2017,40(1):42-47.
[21]赵萍萍,霍仕霞,彭晓明,等.驱虫斑鸠菊中紫铆素、紫铆花素的分离及其对人A375黑素瘤细胞黑素合成作用的影响[J].中国药学杂志,2013,48(20):1724-1727.
[22]赵焕新,白虹,李巍,等.苏木化学成分的研究[J].食品与药品,2010,12(5):176-180.
[23]邵红军,房立真,刘吉开.西澳粘滑菇的化学成分研究[J].天然产物研究与开发,2010,22(5):786-788.
[24]石磊岭,马国需,杨峻山,等.天山假狼毒的化学成分研究[J].中草药,2016,47(2):223-226.
[25]汤俊,SUPINYA T,王峥涛,等.蚬壳花椒茎中的橙黄胡椒酰胺[J].中国药学,2003,12(4):231-233.
[26]刘世杰.毛支清口服液的质量控制、制备工艺及抗炎作用研究[D].北京:中央民族大学,2013.
[27]张怀念,张咏莉.宫颈癌分子遗传机制及其相关基因功能研究[J].安徽医药,2015,19(2):219-221.
[28]李津.氨肽酶N抑制剂与5-FU及其衍生物联合抗肿瘤活性研究[D].济南:山东大学,2015.
[29]MUHAMMAD K.PD-L1单链抗体的筛选、抗体偶联药物的制备以及抗肿瘤活性的研究[D].杭州:浙江大学,2018.
[30]李希哲,黄海燕,赵威,等.桂枝化合物的分离与鉴定及神经保护作用[J].沈阳药科大学学报,2016,33(1):14-19.
[31]谢雁鸣,王连心,王永炎.临床联合用药机制研究的探讨[J].中国中药杂志,2014,39(18):3424-3426.