大青叶化学成分及抗氧化活性研究
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摘要
采用高效液相色谱-大气压化学电离质谱法(HPLC-APCI/MS)和直接进样的电喷雾串联质谱法(ESI-MS~2)分析大青叶乙酸乙酯、正丁醇和95%乙醇提取物的化学成分。采用Kromasil C18分析柱(4.6 mm×250 mm×5μm),以0.15%乙酸/水-甲醇为流动相进行线性梯度洗脱,流速0.8 mL/min,柱温25℃;采用全波长扫描紫外检测器,检测波长分别为254 nm和265 nm;采用大气压化学电离源和电喷雾电离源,在正、负两种离子模式下进行检测。结果表明,在大青叶乙酸乙酯、正丁醇和95%乙醇提取物中共鉴别出39种化合物,包括生物碱类、有机酸类、核苷嘌呤类、氨基酸类、黄酮类、糖类、卟啉类以及含硫类成分化合物。采用铁离子还原能力法(FRAP)对大青叶的抗氧化活性进行评价,并用自由基清除能力法验证FRAP法的测试结果。抗氧化活性评价结果表明,大青叶抗氧化活性部位主要集中在极性较低的化学组分中,吲哚类、奎唑酮类和有机酸类化合物是其主要有效成分。该方法快捷、灵敏,适用于中药化学成分分析和抗氧化活性评价。
High performance liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry(HPLC-APCI/MS) and electrospray ionization tandem mass spectrometry(ESI-MS~2) were applied to analyze the chemical constituents from acetate, n-BuOH and 95% ethanol extracts of Isatidis Folium. The separation was performed on a Kromasil C18 column(4.6 mm×250 mm×5 μm) with the gradient of 0.15% acetic acid/water-methanol as mobile phase with a flow rate of 0.8 mL/min at 25 ℃. The UV spectra were obtained by a diode array detector(DAD) under the detection wavelength at 254 nm and 265 nm. In order to get exact data of MS and MS~2 spectra, the atmosphere pressure chemical ionization ion source(APCI) and electrospray ionization ion source(ESI) in both positive and negative mode were utilized. Up to date, thirty-nine different compounds were identified from the crude extracts, including alkaloids, organic acids, nucleosides and purines, amino acids, flavones, carbohydrates, porphyrin and epigoitrin. Meanwhile, the antioxidant activity of the extracts from Radix Isatidis were successfully estimated by the ferric reducing antioxidant power(FRAP) and radical scavenging(DPPH) methods. The results of the antioxidant activities indicated that the compositions of antioxidant activity were mainly existed in the small polarity part. Indoles, quinazolinones, organic acids and porphyrins might be the main antioxidative constituents of Radix Isatidis. This method is rapid and sensitive that can be used for analyzing the chemical compositions and antioxidant activities of traditional Chinese medicine.
High performance liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry(HPLC-APCI/MS) and electrospray ionization tandem mass spectrometry(ESI-MS~2) were applied to analyze the chemical constituents from acetate, n-BuOH and 95% ethanol extracts of Isatidis Folium. The separation was performed on a Kromasil C18 column(4.6 mm×250 mm×5 μm) with the gradient of 0.15% acetic acid/water-methanol as mobile phase with a flow rate of 0.8 mL/min at 25 ℃. The UV spectra were obtained by a diode array detector(DAD) under the detection wavelength at 254 nm and 265 nm. In order to get exact data of MS and MS~2 spectra, the atmosphere pressure chemical ionization ion source(APCI) and electrospray ionization ion source(ESI) in both positive and negative mode were utilized. Up to date, thirty-nine different compounds were identified from the crude extracts, including alkaloids, organic acids, nucleosides and purines, amino acids, flavones, carbohydrates, porphyrin and epigoitrin. Meanwhile, the antioxidant activity of the extracts from Radix Isatidis were successfully estimated by the ferric reducing antioxidant power(FRAP) and radical scavenging(DPPH) methods. The results of the antioxidant activities indicated that the compositions of antioxidant activity were mainly existed in the small polarity part. Indoles, quinazolinones, organic acids and porphyrins might be the main antioxidative constituents of Radix Isatidis. This method is rapid and sensitive that can be used for analyzing the chemical compositions and antioxidant activities of traditional Chinese medicine.
引文
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[7] 陈瀚,李进,李祥,陈建伟,薛平.板蓝根不同提取部位的体外抗氧化活性[J].中国实验方剂学杂志,2012,18(9):184-186.CHEN Han,LI Jin,LI Xiang,CHEN Jianwei,XUE Ping.Antioxidation of different extracts from Isatidis indigotica in vitro[J].Chinese Journal of Experimental Traditional Medical Formulae,2012,18(9):184-186(in Chinese).
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[11] FERRERS F,ANDRADE P B,VALENTAO P,GIL-IZQUIERDO A.Further knowledge on barely (Hordeum vulgare L.) leaves O-glycosyl-C-glycosyl flavones by liquid chromatography-UV diode-array detection-electrospray ionization mass spectrometry[J].Journal of Chromatography A,2008,1 182(1):56-64.
[12] FERRERS F,GIL-IZQUIERDO A,ANDRADE P B,VALENTAO P,TOMAS-BARBERAN F A.Characterization of C-glycosyl flavones O-glycosylated by liquid chromatography-tandem mass spectrometry[J].Journal of Chromatography A,2007,1 161:214-223.
[13] 刘舒,闫峻,李慧琳,宋凤瑞,刘忠英,刘志强,刘淑莹.复方板蓝根颗粒化学成分的质谱研究[J].高等学校化学学报,2010,31(6):1 143-1 147.LIU Shu,YAN Jun,LI Huilin,SONG Fengrui,LIU Zhongying,LIU Zhiqiang,LIU Shuying.Studies on chemical constituents of compound Indigowoad Root Granule by mass spectrometry[J].Chemical Journal of Chinese Universities,2010,31(6):1 143-1 147(in Chinese).
[14] LU W Y,KIMBALL E,RABINOWITZ J D.A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites[J].Journal of the American Society for Mass Spectrometry,2006,17(1):37-50.
[15] 李锐,付铁军,及元乔,丁立生,彭树林.膜荚黄芪与蒙古黄芪化学成分的高效液相色谱-质谱研究[J].分析化学,2005,33(12):1 676-1 680.LI Rui,FU Tiejun,JI Yuanqiao,DING Li-sheng,PENG Shulin.A study of a Stragalus Mongholicus and a stragalus Menbranaceus by high performance liquid chromatography-mass spectrometry[J].Chinese Journal of Analytical Chemistry,2005,33(12):1 676-1 680(in Chinese).
[16] 郭明全,宋凤瑞,陈茂连,白玉,刘志强,刘淑莹.刺五加寡糖的电喷雾多级串联质谱研究[J].高等学校化学学报,2003,24(9):1 577-1 581.GUO Mingquan,SONG Fengrui,CHEN Mao-lian,BAI Yu,LIU Zhiqiang,LIU Shuying.Studies on oligosaccharides from Acanthopanax Senticosus Harms (AS) by electrospray ionization tandem mass spectrometry (MSn)[J].Chemical Journal of Chinese Universities,2003,24(9):1 577-1 581(in Chinese).
[17] 刘巍,毛友安,钟科军,卢红兵.直接电喷雾离子阱质谱法快速表征烟叶[J].分析试验室,2008,27(2):65-69.LIU Wei,MAO You’an,ZHONG Kejun,LU Hongbing.Fast characterization of tobacco leaves by direct electrospray ionization-ion trap mass spectrometry[J].Chinese Journal of Analysis Laboratory,2008,27(2):65-69(in Chinese).
[18] 闫峻,冯硕,吴韬,LIU R H,TSAO R.亚麻子发芽前后化学成分及抗氧化活性的研究[J].质谱学报,2018,39(1):112-119.YAN Jun,FENG Shuo,WU Tao,LIU Ronghua,TSAO Rong.Chemical constituents and antioxidant activity of flaxseed during germination[J].Journal of Chinese Mass Spectrometry Society,2018,39(1):112-119(in Chinese).
[2] 武彦文,高文远,肖小河.大青叶的研究进展[J].中草药,2006,37(5):793-796.WU Yanwen,GAO Wenyuan,XIAO Xiaohe.Advances in studies on Folium Isatidis[J].Chinese Traditional and Herbal Drugs,2006,37(5):793-796(in Chinese).
[3] 赵震毅,李国斌.异金雀花素等5种大青叶黄酮的同时检测[J].食品工业,2017,38(10):283-286.ZHAO Zhenyi,LI Guobin.Determination of five flavonoids in different Folium Isatidis extracts by using HPLC[J].The Food Industry,2017,38(10):283-286(in Chinese).
[4] 闫峻,赵春芳,李伯平,刘志强,郭冬发,郑重.板蓝根化学成分及抗氧化活性的研究[J].质谱学报,2017,38(2):248-255.YAN Jun,ZHAO Chunfang,LI Boping,LIU Zhiqiang,GUO Dongfa,ZHENG Zhong.Chemical constituents and antioxidant activity of Radix Isatidis[J].Journal of Chinese Mass Spectrometry Society,2017,38(2):248-255(in Chinese).
[5] 闫峻,刘舒,李伯平,刘志强,郭冬发.复方板蓝根颗粒APCI-MS指纹图谱及化学成分的LC-APCI-MSn研究[J].质谱学报,2017,38(3):320-327.YAN Jun,LIU Shu,LI Boping,LIU Zhiqiang,GUO Dongfa.Fingerprint chromatograms and chemical components of compound indigowoad granule by APCI-MS and LC-APCI-MSn[J].Journal of Chinese Mass Spectrometry Society,2017,38(3):320-327(in Chinese).
[6] 孙丽萍,王大仟,张智武.11种天然植物提取物对DPPH自由基的清除作用[J].食品科学,2009,30(1):45-47.SUN Liping,WANG Daqian,ZHANG Zhiwu.2,2-Dipheny-1-picrylhydrazyl Radical Scavenging activites of eleven spenies of natural plant extracts[J].Food Science,2009,30(1):45-47(in Chinese).
[7] 陈瀚,李进,李祥,陈建伟,薛平.板蓝根不同提取部位的体外抗氧化活性[J].中国实验方剂学杂志,2012,18(9):184-186.CHEN Han,LI Jin,LI Xiang,CHEN Jianwei,XUE Ping.Antioxidation of different extracts from Isatidis indigotica in vitro[J].Chinese Journal of Experimental Traditional Medical Formulae,2012,18(9):184-186(in Chinese).
[8] MHON T,PLITZKO I,HAMBURGER M.A comprehensive metabolite profiling of Isatis tinctoria leaf extracts[J].Phytochemistry,2009,70(7):924-934.
[9] XIAO S S,BI K S,SUN Y Q.Identification of chemical constituents in the root of Isatis Indigotica Fort.by LC/DAD/ESI/MS/MS[J].Journal of Liquid Chromatography,2007,30(1):73-85.
[10] DENG X Y,GAO G H,ZHENG S N,LI F M.Qualitative and quantitative analysis of flavonoids in the leaves of Isatis indigatica Fort.by ultra-performance liquid chromatography with PDA and electrospray ionization tandem mass spectrometry detection[J].Journal of Pharmaceutical and Biomedical Analysis,2008,48(3):562-567.
[11] FERRERS F,ANDRADE P B,VALENTAO P,GIL-IZQUIERDO A.Further knowledge on barely (Hordeum vulgare L.) leaves O-glycosyl-C-glycosyl flavones by liquid chromatography-UV diode-array detection-electrospray ionization mass spectrometry[J].Journal of Chromatography A,2008,1 182(1):56-64.
[12] FERRERS F,GIL-IZQUIERDO A,ANDRADE P B,VALENTAO P,TOMAS-BARBERAN F A.Characterization of C-glycosyl flavones O-glycosylated by liquid chromatography-tandem mass spectrometry[J].Journal of Chromatography A,2007,1 161:214-223.
[13] 刘舒,闫峻,李慧琳,宋凤瑞,刘忠英,刘志强,刘淑莹.复方板蓝根颗粒化学成分的质谱研究[J].高等学校化学学报,2010,31(6):1 143-1 147.LIU Shu,YAN Jun,LI Huilin,SONG Fengrui,LIU Zhongying,LIU Zhiqiang,LIU Shuying.Studies on chemical constituents of compound Indigowoad Root Granule by mass spectrometry[J].Chemical Journal of Chinese Universities,2010,31(6):1 143-1 147(in Chinese).
[14] LU W Y,KIMBALL E,RABINOWITZ J D.A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites[J].Journal of the American Society for Mass Spectrometry,2006,17(1):37-50.
[15] 李锐,付铁军,及元乔,丁立生,彭树林.膜荚黄芪与蒙古黄芪化学成分的高效液相色谱-质谱研究[J].分析化学,2005,33(12):1 676-1 680.LI Rui,FU Tiejun,JI Yuanqiao,DING Li-sheng,PENG Shulin.A study of a Stragalus Mongholicus and a stragalus Menbranaceus by high performance liquid chromatography-mass spectrometry[J].Chinese Journal of Analytical Chemistry,2005,33(12):1 676-1 680(in Chinese).
[16] 郭明全,宋凤瑞,陈茂连,白玉,刘志强,刘淑莹.刺五加寡糖的电喷雾多级串联质谱研究[J].高等学校化学学报,2003,24(9):1 577-1 581.GUO Mingquan,SONG Fengrui,CHEN Mao-lian,BAI Yu,LIU Zhiqiang,LIU Shuying.Studies on oligosaccharides from Acanthopanax Senticosus Harms (AS) by electrospray ionization tandem mass spectrometry (MSn)[J].Chemical Journal of Chinese Universities,2003,24(9):1 577-1 581(in Chinese).
[17] 刘巍,毛友安,钟科军,卢红兵.直接电喷雾离子阱质谱法快速表征烟叶[J].分析试验室,2008,27(2):65-69.LIU Wei,MAO You’an,ZHONG Kejun,LU Hongbing.Fast characterization of tobacco leaves by direct electrospray ionization-ion trap mass spectrometry[J].Chinese Journal of Analysis Laboratory,2008,27(2):65-69(in Chinese).
[18] 闫峻,冯硕,吴韬,LIU R H,TSAO R.亚麻子发芽前后化学成分及抗氧化活性的研究[J].质谱学报,2018,39(1):112-119.YAN Jun,FENG Shuo,WU Tao,LIU Ronghua,TSAO Rong.Chemical constituents and antioxidant activity of flaxseed during germination[J].Journal of Chinese Mass Spectrometry Society,2018,39(1):112-119(in Chinese).