超高效液相色谱-四极杆-飞行时间质谱法分析葫芦巴成分
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摘要
采用超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF MS)对葫芦巴的醇提组分和除糖后的水提组分进行定性分析,并以甘草苷为内标,对鉴定出的成分进行半定量分析。采用Unitary C18色谱柱(4.6 mm×150 mm×5μm),以乙腈-0.1%甲酸/水为流动相进行线性梯度洗脱,流速0.5 mL/min,电喷雾离子源(ESI)负离子模式检测。定性分析结果表明,葫芦巴的醇提组分和水提组分的总离子流图相似,共鉴定出36种化合物,包括12种黄酮类成分和24种皂苷类成分。实验选择了几种代表性的黄酮类和皂苷类化合物,对其结构进行详细阐述,并推断其可能的质谱裂解规律。半定量分析结果表明,两种提取物中主成分的相对含量不同,黄酮类化合物在乙醇提取物中的相对含量之和是水提物中的1.13倍,皂苷类化合物在乙醇提取物中的相对含量之和是水提物中的1.32倍。该方法简便、快捷、灵敏,可为葫芦巴的药效物质基础研究和开发应用提供理论依据。
Fenugreek in invigorating kidney, falling blood sugar, etc, have very good medicinal value. Much attention has been paid to saponins, flavonoids, the separation and purification, the compatibility mechanism of these components have been studied in depth, but the preparation is complicated, time-consuming, labor-intensive and poor economic. In this research, compounds in the ethanol extract and the water extract after sugar removal of fenugreek were qualitatively analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOFMS). And the liquiritin was selected as the internal standard for semi-quantitative analysis of all the identified components. Chromatographic separation was performed on a Unitary C18 column(4.6 mm×150 mm×5 μm). Acetonitrile and 0.1%(volume fraction) formic acid solution were used as the mobile phase for gradient elution with the flow rate of 0.5 mL/min. The MS analysis was carried out by the electrospray ionization(ESI) source in negative mode. The qualitative analysis results showed that the total ion chromatograms of the ethanol extract and the water extract after sugar removal of fenugreek were similar with each other, and 36 compounds were identified, including 12 flavonoids and 24 saponin components. Taking typical flavonoids and saponins as examples, the structures of flavonoids and saponin were described in detail, and the possible breaking rules of flavonoids and saponin were deduced. The results of semi-quantitative analysis showed that the relative content of the main components in the two extracts was different. The sum of the relative contents of the flavonoids in the ethanol extract was 1.13 times that in the aqueous extract. The sum of the relative contents of the saponin compounds was 1.32 times that in the aqueous extract. The method is very simple, rapid and sensitive, and the obtained results will provide a theory basis for further research on fenugreek, as well as development and application research of fenugreek.
Fenugreek in invigorating kidney, falling blood sugar, etc, have very good medicinal value. Much attention has been paid to saponins, flavonoids, the separation and purification, the compatibility mechanism of these components have been studied in depth, but the preparation is complicated, time-consuming, labor-intensive and poor economic. In this research, compounds in the ethanol extract and the water extract after sugar removal of fenugreek were qualitatively analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOFMS). And the liquiritin was selected as the internal standard for semi-quantitative analysis of all the identified components. Chromatographic separation was performed on a Unitary C18 column(4.6 mm×150 mm×5 μm). Acetonitrile and 0.1%(volume fraction) formic acid solution were used as the mobile phase for gradient elution with the flow rate of 0.5 mL/min. The MS analysis was carried out by the electrospray ionization(ESI) source in negative mode. The qualitative analysis results showed that the total ion chromatograms of the ethanol extract and the water extract after sugar removal of fenugreek were similar with each other, and 36 compounds were identified, including 12 flavonoids and 24 saponin components. Taking typical flavonoids and saponins as examples, the structures of flavonoids and saponin were described in detail, and the possible breaking rules of flavonoids and saponin were deduced. The results of semi-quantitative analysis showed that the relative content of the main components in the two extracts was different. The sum of the relative contents of the flavonoids in the ethanol extract was 1.13 times that in the aqueous extract. The sum of the relative contents of the saponin compounds was 1.32 times that in the aqueous extract. The method is very simple, rapid and sensitive, and the obtained results will provide a theory basis for further research on fenugreek, as well as development and application research of fenugreek.
引文
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[7] 周慧,宋凤瑞,刘志强,郑毅男,刘淑莹.刺五加叶的HPLC-UV和ESI-MS指纹图谱研究[J].质谱学报,2008,29(6):321-326.ZHOU Hui,SONG Fengrui,LIU Zhiqiang,ZHENG Yinan,LIU Shuying.The fingerprints of leaves of Acanthopanax Senticosus by HPLC-UV and ESI-MS[J].Journal of Chinese Mass Spectrometry Society,2008,29(6):321-326(in Chinese).
[8] 杨梅,朱家勇,颜克序,殷岚,段秀梅.葫芦巴多糖提取工艺的研究[J].广东药学院学报,2014,30(04):444-447.YANG Mei,ZHU Jiayong,YAN Kexu,YIN Lan,DUAN Xiumei.Study on the extraction process of polysaccharides from Fenugreek[J].Journal of Guangdong Pharmaceutical University,2014,30(04):444-447(in Chinese).
[9] 邵明昱.葫芦巴种子总黄酮提取分离、抑菌及抗氧化活性的研究[D].乌鲁木齐:新疆农业大学,2012.
[10] 黑晶,陈挚,雷亚亚,王文苹,隋宏.葫芦巴总皂苷的提取纯化工艺考察[J].中国实验方剂学杂志,2014,20(5):11-13.HEI Jing,CHEN Zhi,LEI Yaya,WANG Wenping,SUI Hong.Investigation of extraction and purification process for total saponins in trigonellae semen[J].Chinese Journal of Experimental Traditional Medical Formulae,2014,20(5):11-13(in Chinese).
[11] 汪佳琦.葫芦巴降血糖组分协同增效作用机制研究[D].长春:吉林大学,2017.
[12] 乔善义,郭继芬,赵毅民,张爽.MS/MS技术快速发现和分析繁缕中的黄酮碳苷成分[J].中国天然药物,2003,1(2):120-123.QIAO Shanyi,GUO Jifen,ZHAO Yimin,ZHANG Shuang.Rapid discovery and analysis of C-glycosyflavones from stellaria media (l.) cyr by MS/MS[J].Chinese Journal of Natural Medicines,2003,1(2):120-123(in Chinese).
[13] 吴新安,都模勤.黄酮碳苷类化合物ESI-MS/MS裂解规律初探[J].天然产物研究与开发,2011,23(6):1 085-1 087.WU Xin’an,DOU Moqin.Rule of ESI-MS/MS on C-glycosyflavones[J].Natural Product Research and Development,2011,23(6):1 085-1 087(in Chinese).
[14] FERRERES F,SILVA B M,ANDRADEP B,FERREIRA M A,SEABRA R M.Approach to the study of C-glycosyl flavones by ion trap HPLC-PAD-ESI/MS/MS:applicationto seeds of quince (cydonia oblonga)[J].Phytochemical Analysis,2003,14(6):352-359.
[15] BENAYAD Z,CARMEN G C,Eddine E S.Identification and quantification of flavonoid glycosides from fenugreek (Trigonella foenum-graecum) germinatedseeds by LC-DAD-ESI/MS analysis[J].Journal of Food Composition and Analysis,2014,35(1):21-29.
[16] KROL K B,GLOD D,KRAUZE B M,MAT-?AWSKA I.Application of one- and two-dimensional high-performance liquid chromatography methodologies for the analysis of C-glycosylfavones from fenugreek seeds[J].Journal of Chromatography A,2014,1 367:48-56.
[17] BENAYAD Z,CARMEN G C,Eddine E S.Characterization of flavonoid glycosides from fenugreek (Trigonella foenum-graecum) crude seeds by HPLC-DAD-ESI/MS analysis[J].International Journal of Molecular Sciences,2014,15(11):20 668-20 685.
[18] RAYYAN S,FOSSEN T,ANDERSEN M.Flavone C-glycosides from seeds of fenugreek,Trigonella foenum-graecuml[J].Journal of Agricultural and Food Chemistry,2010,58(12):7 211-7 217.
[19] KANG L P,ZHAO Y,PANG X,YU H S,XIONG C Q,ZHANG J,GAO Y,YU K,LIU C,MA B P.Characterization and identification of steroidal saponins from the seeds of Trigonella foenum-graecum by ultra high performance liquid chromatography and hybrid time-of-flight mass spectrometry[J].Journal of Pharmaceutical and Biomedical Analysis,2013,74:257-267.
[20] PANG X,KANG L,YU H,ZHAO Y,XIONG C,ZHANG J,SHAN J,MA B.Rapid isolation of new furostanol saponins from febnugreek seeds based on ultra-performance liquid chromatography coupled with a hybrid quadrupole time-of-flight tandem mass spectrometry[J].Journal of Separation Science,2012,35(12):1 538-1 550.
[21] GUPTA R K,JAIN D C,THAKUR R S.Furostanol glycosides from Trigonella foeum-graecum seeds[J].Phytochemistry,1 984,23(11):2 605-2 607.
[2] 王珊,王娜,王萍,齐富友,董爱君.葫芦巴浸膏挥发性成分分析[J].香料香精化妆品,2016,10(5):10-12.WANG Shan,WANG Na,WANG Ping,QI Fuyou,DONG Aijun.Analysis of volatile components in fenugreek concrete[J].Flavour Fragrance Cosmetics,2016,10(5):10-12(in Chinese).
[3] 丁建海,马晓芳,俞剑,刘世巍,张俊芳.HPLC法测定葫芦巴中薯蓣皂苷元的含量[J].广州化工,2015,43(14):118-119.DING Jianhai,MA Xiaofang,YU Jian,LIU Shiwei,ZHANG Junfang.Determination of the contents of diosigenin of the Trionella foenum-graecum by HPLC[J].Guangzhou Chemical Industry,2015,43(14):118-119(in Chinese).
[4] 丁建海,牛浩军,刘世巍,张方娟.分光光度法测定葫芦巴中总黄酮含量[J].广州化工,2017,45(9):125-127.DING Jianhai,NIU Haojun,LIU Shiwei,ZHANG Fangjuan.Total flavonoids determination of foenum-graecum by ultraviolet spectrophotometry[J].Guangzhou Chemical Industry,2017,45(9):125-127(in Chinese).
[5] 叶荣飞,程侠,宋森川,李峰,黄飞,任成龙,宋化灿.在线热裂解-气相色谱/质谱联用技术分析葫芦巴净油的热裂解产物[J].中山大学学报:自然科学版,2015,54(2):83-88.YE Rongfei,CHENG Xia,SONG Senchuan,LI Feng,HUANG Fei,REN Chenglong,SONG Huacan.Analysis of pyrolysates from fenugreek absolute by on-line pyrolysis-gas chromatography/mass spectrometry[J].Acta Scientiarum Naturalium Universitatis Sunyatsen,2015,54(2):83-88(in Chinese).
[6] 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).
[7] 周慧,宋凤瑞,刘志强,郑毅男,刘淑莹.刺五加叶的HPLC-UV和ESI-MS指纹图谱研究[J].质谱学报,2008,29(6):321-326.ZHOU Hui,SONG Fengrui,LIU Zhiqiang,ZHENG Yinan,LIU Shuying.The fingerprints of leaves of Acanthopanax Senticosus by HPLC-UV and ESI-MS[J].Journal of Chinese Mass Spectrometry Society,2008,29(6):321-326(in Chinese).
[8] 杨梅,朱家勇,颜克序,殷岚,段秀梅.葫芦巴多糖提取工艺的研究[J].广东药学院学报,2014,30(04):444-447.YANG Mei,ZHU Jiayong,YAN Kexu,YIN Lan,DUAN Xiumei.Study on the extraction process of polysaccharides from Fenugreek[J].Journal of Guangdong Pharmaceutical University,2014,30(04):444-447(in Chinese).
[9] 邵明昱.葫芦巴种子总黄酮提取分离、抑菌及抗氧化活性的研究[D].乌鲁木齐:新疆农业大学,2012.
[10] 黑晶,陈挚,雷亚亚,王文苹,隋宏.葫芦巴总皂苷的提取纯化工艺考察[J].中国实验方剂学杂志,2014,20(5):11-13.HEI Jing,CHEN Zhi,LEI Yaya,WANG Wenping,SUI Hong.Investigation of extraction and purification process for total saponins in trigonellae semen[J].Chinese Journal of Experimental Traditional Medical Formulae,2014,20(5):11-13(in Chinese).
[11] 汪佳琦.葫芦巴降血糖组分协同增效作用机制研究[D].长春:吉林大学,2017.
[12] 乔善义,郭继芬,赵毅民,张爽.MS/MS技术快速发现和分析繁缕中的黄酮碳苷成分[J].中国天然药物,2003,1(2):120-123.QIAO Shanyi,GUO Jifen,ZHAO Yimin,ZHANG Shuang.Rapid discovery and analysis of C-glycosyflavones from stellaria media (l.) cyr by MS/MS[J].Chinese Journal of Natural Medicines,2003,1(2):120-123(in Chinese).
[13] 吴新安,都模勤.黄酮碳苷类化合物ESI-MS/MS裂解规律初探[J].天然产物研究与开发,2011,23(6):1 085-1 087.WU Xin’an,DOU Moqin.Rule of ESI-MS/MS on C-glycosyflavones[J].Natural Product Research and Development,2011,23(6):1 085-1 087(in Chinese).
[14] FERRERES F,SILVA B M,ANDRADEP B,FERREIRA M A,SEABRA R M.Approach to the study of C-glycosyl flavones by ion trap HPLC-PAD-ESI/MS/MS:applicationto seeds of quince (cydonia oblonga)[J].Phytochemical Analysis,2003,14(6):352-359.
[15] BENAYAD Z,CARMEN G C,Eddine E S.Identification and quantification of flavonoid glycosides from fenugreek (Trigonella foenum-graecum) germinatedseeds by LC-DAD-ESI/MS analysis[J].Journal of Food Composition and Analysis,2014,35(1):21-29.
[16] KROL K B,GLOD D,KRAUZE B M,MAT-?AWSKA I.Application of one- and two-dimensional high-performance liquid chromatography methodologies for the analysis of C-glycosylfavones from fenugreek seeds[J].Journal of Chromatography A,2014,1 367:48-56.
[17] BENAYAD Z,CARMEN G C,Eddine E S.Characterization of flavonoid glycosides from fenugreek (Trigonella foenum-graecum) crude seeds by HPLC-DAD-ESI/MS analysis[J].International Journal of Molecular Sciences,2014,15(11):20 668-20 685.
[18] RAYYAN S,FOSSEN T,ANDERSEN M.Flavone C-glycosides from seeds of fenugreek,Trigonella foenum-graecuml[J].Journal of Agricultural and Food Chemistry,2010,58(12):7 211-7 217.
[19] KANG L P,ZHAO Y,PANG X,YU H S,XIONG C Q,ZHANG J,GAO Y,YU K,LIU C,MA B P.Characterization and identification of steroidal saponins from the seeds of Trigonella foenum-graecum by ultra high performance liquid chromatography and hybrid time-of-flight mass spectrometry[J].Journal of Pharmaceutical and Biomedical Analysis,2013,74:257-267.
[20] PANG X,KANG L,YU H,ZHAO Y,XIONG C,ZHANG J,SHAN J,MA B.Rapid isolation of new furostanol saponins from febnugreek seeds based on ultra-performance liquid chromatography coupled with a hybrid quadrupole time-of-flight tandem mass spectrometry[J].Journal of Separation Science,2012,35(12):1 538-1 550.
[21] GUPTA R K,JAIN D C,THAKUR R S.Furostanol glycosides from Trigonella foeum-graecum seeds[J].Phytochemistry,1 984,23(11):2 605-2 607.