聚酰胺色谱法联合中压液相制备色谱法提取分离镰形棘豆中活性黄酮类成分
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摘要
目的:对藏药镰形棘豆中黄酮类成分进行提取分离,并将得到的黄酮化合物进行抗人肺腺癌A549细胞的活性筛选。方法:镰形棘豆药材浸提液经乙酸乙酯萃取,该萃取部位通过聚酰胺柱色谱梯度洗脱,结合中压液相制备色谱法进一步纯化分离,获得单一黄酮化合物,通过高效液相色谱法(HPLC)、质谱法(MS)及核磁共振波谱法(NMR)对化合物进行分析、鉴定;采用CCK-8法,检测黄酮化合物对人肺腺癌A549细胞的抑制作用。结果:从镰形棘豆的乙酸乙酯部位分离得到2个黄酮类单体,鉴定为7-羟基二氢黄酮及2',4'-二羟基二氢查尔酮,HPLC分析两者纯度均大于98%; CCK-8法结果显示,7-羟基二氢黄酮及2',4'-二羟基二氢查尔酮均能显著抑制A549细胞活力,并呈量效关系,48 h的IC50值分别为137. 6μg/m L和63. 83μg/m L。结论:分离得到的2个黄酮成分对A549细胞均有良好的抑制作用,为后期抗肺癌机制研究提供了实验基础。聚酰胺柱色谱法具有富集黄酮的作用,适用于分离黄酮类成分,聚酰胺色谱法联合中压制备液相色谱法,提取分离镰形棘豆中黄酮类成分,简便易行,为分离黄酮类成分提供参考。
Objective: To extract and isolate the flavonoids from Oxytropis falcata( Of),and screen bioactive flavonoids on human lung adenocarcinoma A549 cells in vitro. Methods: Crude extraction was extracted from Of by ethyl acetate. Ethyl acetate extraction was further separated by polyamide column chromatography with gradient elution and purified by medium-pressure liquid chromatography to obtain the flavonoids; High performance liquid chromatography( HPLC),mass spectrometry( MS) and nuclear magnetic resonance spectroscopy( NMR) were used to analyze and elucidate the structure of the obtained flavonoids. CCK-8 analysis was used to detect the inhibited viability of flavonoids on human lung adenocarcinoma A549 cells in vitro. Results: There were 2 flavonoids isolated from Of ethyl acetate fraction,and were identified as 7-hydroxy dihydroflavone and 2',4'-dihydroxy dihydrochalcone. Purity of them were both greater than 98% by HPLC analyses; The results of CCK-8 showed that 7-hydroxy dihydroflavone and 2',4'-dihydroxy dihydrochalcone could significantly inhibit the viability of A549 cells in a dose dependent manner. The IC50 values were 137. 6 μg/mL and 63. 83 μg/mL at 48 h,respectively. Conclusion: The 2 flavonoids have effective bioactivities on inhibiting proliferation of human lung adenocarcinoma A549 cells,which could provide the experimental basis for the further studies on anti-lung cancer mechanism. Polyamide column chromatography is suitable for enrichment and isolation of flavonoids. Polyamide column chromatography combined with medium pressure liquid chromatography is simple and easy to prepare various flavonoids.Thus,the method could provide a reference for obtaining flavonoids from Chinese medicines.
Objective: To extract and isolate the flavonoids from Oxytropis falcata( Of),and screen bioactive flavonoids on human lung adenocarcinoma A549 cells in vitro. Methods: Crude extraction was extracted from Of by ethyl acetate. Ethyl acetate extraction was further separated by polyamide column chromatography with gradient elution and purified by medium-pressure liquid chromatography to obtain the flavonoids; High performance liquid chromatography( HPLC),mass spectrometry( MS) and nuclear magnetic resonance spectroscopy( NMR) were used to analyze and elucidate the structure of the obtained flavonoids. CCK-8 analysis was used to detect the inhibited viability of flavonoids on human lung adenocarcinoma A549 cells in vitro. Results: There were 2 flavonoids isolated from Of ethyl acetate fraction,and were identified as 7-hydroxy dihydroflavone and 2',4'-dihydroxy dihydrochalcone. Purity of them were both greater than 98% by HPLC analyses; The results of CCK-8 showed that 7-hydroxy dihydroflavone and 2',4'-dihydroxy dihydrochalcone could significantly inhibit the viability of A549 cells in a dose dependent manner. The IC50 values were 137. 6 μg/mL and 63. 83 μg/mL at 48 h,respectively. Conclusion: The 2 flavonoids have effective bioactivities on inhibiting proliferation of human lung adenocarcinoma A549 cells,which could provide the experimental basis for the further studies on anti-lung cancer mechanism. Polyamide column chromatography is suitable for enrichment and isolation of flavonoids. Polyamide column chromatography combined with medium pressure liquid chromatography is simple and easy to prepare various flavonoids.Thus,the method could provide a reference for obtaining flavonoids from Chinese medicines.
引文
[1]高博,李钦,杨丽霞,等.镰形棘豆的研究现状与分析[J].中医研究,2017,30(1):75-78.
[2]帝玛尔·丹增彭措.晶珠本草[M].上海:上海科学技术出版社,1986.
[3]中华人民共和国卫生部.中华人民共和国卫生部药品标准:藏药:第1册[S].1995:340.
[4]张晓晶,李凌宇,格桑罗布,等.藏药镰形棘豆中的黄酮类化合物(英文)[J].Journal of Chinese Pharmaceutical Sciences,2014,23(2):99-105.
[5]顾鹏程,许惠琴.镰形棘豆化学成分和药理毒理作用的研究进展[J].时珍国医国药,2009,20(10):2549-2550.
[6]陈锦珊,杨钦磊,刘晓玲,等.藏药镰形棘豆的化学成分、药理作用及毒理学研究进展[J].中国药房,2016,27(28):3945-3.
[7]楼成华,王明艳,杨欢,等.镰形棘豆中黄酮类化合物抗肿瘤活性的体外实验研究[J].南京中医药大学学报,2009,25(1):46-50.
[8]王栋,唐炜,杨光明,等.藏药镰形棘豆黄酮类成分的抗炎、抗氧化及细胞毒性研究(英文)[J].中国天然药物,2010,8(6):461-465.
[9]LI A,SUN A,LIU R,et al.An efficient preparative procedure for main flavonoids from the peel of Trichosanthes kirilowii Maxim.using polyamide resin followed by semi-preparative high performance liquid chromatography[J].Journal of Chromatography B,2014,965:150-157.
[10]LIU GY,ZHUANG LW,SONG DD,et al.Isolation,purification,and identification of the main phenolic compounds from leaves of celery(Apium graveolens L.var.dulce Mill./Pers.)[J].Journal of Separation Science,2017,40:472-479.
[11]李旭升,蒋鑫炜,单晓煜,等.黑豆皮花色苷的中压液相色谱制备及结构鉴定[J].食品与发酵工业,2018,44(6):51-57.
[12]杨欢,王栋,童丽,等.镰形棘豆的化学成分研究(Ⅲ)[J].南京中医药大学学报,2008,24(1):43-44.
[13]吕芳,徐筱杰.藏药镰形棘豆中黄酮类化学成分研究[J].中国中药杂志,2007,32(4):318-320.
[14]确生.藏药镰形棘豆化学成分的研究[D].兰州:西北师范大学,2003.
[15]魏学红,金莉.藏药镰形棘豆的研究进展[J].中国药理学通报,2010,26(11):1535-1538.
[16]杨光明,燕珂,顾青,等.藏药镰形棘豆对SMMC-7721肝癌细胞凋亡及Bcl-2,Bax蛋白表达的影响[J].中国药学杂志,2013,48(24):2113-2116.
[17]孙兆林,王晓,王岱杰,等.聚酰胺色谱结合高速逆流色谱分离制备萹蓄黄酮类化合物[J].天然产物研究与开发,2015,27(5):854-8.
[2]帝玛尔·丹增彭措.晶珠本草[M].上海:上海科学技术出版社,1986.
[3]中华人民共和国卫生部.中华人民共和国卫生部药品标准:藏药:第1册[S].1995:340.
[4]张晓晶,李凌宇,格桑罗布,等.藏药镰形棘豆中的黄酮类化合物(英文)[J].Journal of Chinese Pharmaceutical Sciences,2014,23(2):99-105.
[5]顾鹏程,许惠琴.镰形棘豆化学成分和药理毒理作用的研究进展[J].时珍国医国药,2009,20(10):2549-2550.
[6]陈锦珊,杨钦磊,刘晓玲,等.藏药镰形棘豆的化学成分、药理作用及毒理学研究进展[J].中国药房,2016,27(28):3945-3.
[7]楼成华,王明艳,杨欢,等.镰形棘豆中黄酮类化合物抗肿瘤活性的体外实验研究[J].南京中医药大学学报,2009,25(1):46-50.
[8]王栋,唐炜,杨光明,等.藏药镰形棘豆黄酮类成分的抗炎、抗氧化及细胞毒性研究(英文)[J].中国天然药物,2010,8(6):461-465.
[9]LI A,SUN A,LIU R,et al.An efficient preparative procedure for main flavonoids from the peel of Trichosanthes kirilowii Maxim.using polyamide resin followed by semi-preparative high performance liquid chromatography[J].Journal of Chromatography B,2014,965:150-157.
[10]LIU GY,ZHUANG LW,SONG DD,et al.Isolation,purification,and identification of the main phenolic compounds from leaves of celery(Apium graveolens L.var.dulce Mill./Pers.)[J].Journal of Separation Science,2017,40:472-479.
[11]李旭升,蒋鑫炜,单晓煜,等.黑豆皮花色苷的中压液相色谱制备及结构鉴定[J].食品与发酵工业,2018,44(6):51-57.
[12]杨欢,王栋,童丽,等.镰形棘豆的化学成分研究(Ⅲ)[J].南京中医药大学学报,2008,24(1):43-44.
[13]吕芳,徐筱杰.藏药镰形棘豆中黄酮类化学成分研究[J].中国中药杂志,2007,32(4):318-320.
[14]确生.藏药镰形棘豆化学成分的研究[D].兰州:西北师范大学,2003.
[15]魏学红,金莉.藏药镰形棘豆的研究进展[J].中国药理学通报,2010,26(11):1535-1538.
[16]杨光明,燕珂,顾青,等.藏药镰形棘豆对SMMC-7721肝癌细胞凋亡及Bcl-2,Bax蛋白表达的影响[J].中国药学杂志,2013,48(24):2113-2116.
[17]孙兆林,王晓,王岱杰,等.聚酰胺色谱结合高速逆流色谱分离制备萹蓄黄酮类化合物[J].天然产物研究与开发,2015,27(5):854-8.