HPLC-DPPH·-UV/Vis-MS法筛选窄叶鲜卑花叶提取物中的抗氧化活性成分
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摘要
目的:利用HPLC-DPPH·-UV/Vis-MS技术筛选并鉴定窄叶鲜卑花叶中的抗氧化活性成分,并比较不同产地的差异。方法:窄叶鲜卑花叶提取物通过Agilent 1260液相色谱系统进行分离,流动相为乙腈-0.2%甲酸水溶液,梯度洗脱,检测波长为325 nm;DPPH·反应液用岛津SPD~(-1)0Avp系统输送,检测波长为517 nm。通过单因素试验优化抗氧化活性物质的筛选方法。利用电喷雾飞行时间质谱(负离子模式),得到各个组分的结构信息,并结合对照品及相关文献进行鉴定。结果:在线衍生的最佳条件为DPPH·甲醇溶液,浓度为30μg·m L~(-1),流速为0.3 m L·min~(-1),PEEK反应管(0.254 mm)长度为11 m。从窄叶鲜卑花叶中筛选出14个抗氧化活性物质,其中7个成分得到鉴定,10个为不同产地样品共有成分,青海西宁市郊和祁连县样品含较为丰富的抗氧化成分。结论:该筛选方法快速、准确、重现性好,适用于窄叶鲜卑花叶中抗氧化活性物质的筛选。
Objective:To screen and identify the antioxidant components in Sibiraea angustata leaves using the HPLC-DPPH·-UV/Vis-MS method,and to compare the similarities and differences of the antioxidant compounds in different samples from different habitants. Methods:The sample was separated by Agilent 1260 HPLC system. The analysis was conducted with acetonitrile and 0.2% aqueous formic acid as mobile phases with gradient elution and the detection wavelength was at 325 nm. The reaction solution of DPPH· was transmitted by SHIMADZU SPD-10 Avp system with the detection wavelength at 517 nm. The screening method for antioxidant substances was optimized by single factor experiment. The structural information was obtained by time of flightmass spectrometry(TOF-MS)with electro-spray ionization(ESI)in negative ion mode. The unknown compounds were identified by reference substance and literature data. Results:The optimal result was conducted with the concentration of DPPH·-methanol solution of 30 μg·mL~(-1),the flow rate of 0.3 mL·min~(-1) and the length of PEEK reactor(0.254 mm)of 11 m. 14 kinds of antioxidant components were screened and identified from Sibiraea angustata leaves. In these compounds,7 of them were identified clearly while 10 of them were common peaks. More abundant antioxidant components were contained in the samples from suburb of Xining and Qilian. Conclusion:The method is rapid,accurate and reproducible,which is suitable for the screening of antioxidant components in the leaves of Sibiraea angustata.
Objective:To screen and identify the antioxidant components in Sibiraea angustata leaves using the HPLC-DPPH·-UV/Vis-MS method,and to compare the similarities and differences of the antioxidant compounds in different samples from different habitants. Methods:The sample was separated by Agilent 1260 HPLC system. The analysis was conducted with acetonitrile and 0.2% aqueous formic acid as mobile phases with gradient elution and the detection wavelength was at 325 nm. The reaction solution of DPPH· was transmitted by SHIMADZU SPD-10 Avp system with the detection wavelength at 517 nm. The screening method for antioxidant substances was optimized by single factor experiment. The structural information was obtained by time of flightmass spectrometry(TOF-MS)with electro-spray ionization(ESI)in negative ion mode. The unknown compounds were identified by reference substance and literature data. Results:The optimal result was conducted with the concentration of DPPH·-methanol solution of 30 μg·mL~(-1),the flow rate of 0.3 mL·min~(-1) and the length of PEEK reactor(0.254 mm)of 11 m. 14 kinds of antioxidant components were screened and identified from Sibiraea angustata leaves. In these compounds,7 of them were identified clearly while 10 of them were common peaks. More abundant antioxidant components were contained in the samples from suburb of Xining and Qilian. Conclusion:The method is rapid,accurate and reproducible,which is suitable for the screening of antioxidant components in the leaves of Sibiraea angustata.
引文
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[13]卫阳飞,刘东花,张宏曦,等.窄叶鲜卑花叶中黄酮和多酚的超声提取工艺及抗氧化性研究[J].中药材,2017,40(1):158WEI YF,LIU DH,ZHANG HX,et al.Ultrasound-assisted extraction technology and antioxidant activity of flavonoids and polyphenols in Sibiraea angustata leaves[J].J Chin Med Mater,2017,40(1):158
[14]陈璐,陶婷婷,王天志.RP-HPLC测定藏药柳茶中阿魏酸和异阿魏酸的含量[J].华西药学杂志,2006,21(2):196CHEN L,TAO TT,WANG TZ.Determination of ferulic acid and isoferulic acid in Sibiraea angustata by RP-HPLC[J].West China J Pharm Sci,2006,21(2):196
[15]闫志慧,陈立书,朱仝飞,等.藏药柳茶叶乙醇水提取物中化学成分的鉴定[J].应用化学,2014,31(2):237YAN ZH,CHEN LS,ZHU TF,et al.Determination of the chemical constituents in aqueous alcohol extract of Sibiraea angustata(Rehd.)Hand-Mazz.leaf[J].Chin J Appl Chem,2014,31(2):237
[16]LI B,CHEN XT,WANG ZW,et al.Two new monoterpenes and one dicaffeic acid ester from Sibiraea angustata with hypolipidemic activities in Hep G2 cells in vitro[J].Phytochem lett,2015,13(7):319
[2]PEDAN V,FISCHER N,ROHN S,et al.An online NP-HPLCDPPH method for the determination of the antioxidant activity of condensed polyphenols in cocoa[J].Food Res Int,2016,89(3):890
[3]ZHANG XX,LIN ZT,FANG JG,et al.An on-line high-performance liquid chromatography-diode-array-detector-electrospray ionizationion-trap-time-of-flight-mass spectrometry total antioxidant capacity detection system applying two antioxidant methods for activity evaluation of the edible flowers from Prunus mume[J].J Chromatogr A,2015,1414(1):88
[4]甘肃省卫生局.甘肃中草药手册.第3册[M].兰州:甘肃人民出版社,1973:1554The Gansu Board of Health.Handbook of Chinese Herbal Medicines in Gansu.Vol 3[M].Lanzhou:Gansu People’s Publishing House,1973:1554
[5]赵汝能.甘肃中草药资源志[M].兰州:甘肃科学技术出版社,2004:31ZHAO RN.The Gansu Herbal Medicine Resource Records[M].Lanzhou:Gansu Science and Technology Press,2004:31
[6]夏苗,王晶宇,马晓燕,等.藏药柳茶提取物调节肥胖大鼠脂代谢的作用机制研究[J].中药材,2011,34(6):922XIA M,WANG JY,MA XY,et al.Effects and mechanism of Sibiraea angustata on lipid matabolism in high-fatted rats[J].Chin Med Mat,2011,34(6):922
[7]姚莉,鞠洋.窄叶鲜卑花促消化作用的实验研究[J].中国中西医结合消化杂志,2009,17(6):376YAO L,JU Y.Effects of Sibiraea angustata on digestive system[J].Chin J Integr Trad West Med Dig,2009,17(6):376
[8]刘昕,潘兴斌,李兴玉.藏药柳茶提取物对K562肿瘤细胞生长的影响[J].中国病理生理杂志,2003,19(2):253LIU X,PAN XB,LI XY.Effect of Liucha extract on tumor K562 cell growth[J].Chin J Pathophysiol,2003,19(2):253
[9]姚莉,骆勤,李红卫,等.柳茶对D-半乳糖胺肝损伤的保护作用及机制[J].中国医院药学杂志,2010,30(6):483YAO L,LUO Q,LI HW,et al.Protective actions of Liu Tea on D-galactosamine induced acute liver injury in rats and its machanism of action[J].Chin Hosp Pharm J,2010,30(6):483
[10]段博文,李运,刘昕,等.柳茶多糖对小鼠免疫功能的影响[J].中国中药杂志,2010,35(11):1466DUAN BW,LI Y,LIU X,et al.Effect of polysaccharides in processed Sibiraea on immunologic function of immunosuppression mice[J].Chin J Chin Mater Med,2010,35(11):1466
[11]武燕,何华,刘健.藏药柳茶挥发油的抗氧化及抑菌活性[J].中国医院药学杂志,2012,32(22):1819WU Y,HE H,LIU J.Antimicrobial and antioxidant properties of the essential oil of Sibiraea angustata RCHD[J].Chin Hosp Pharm J,2012,32(22):1819
[12]刘昕,潘兴斌,封士兰,等.柳茶对小鼠抗氧化能力及免疫器官影响的实验观察[J].兰州医学院学报,1994,20(3):143LIU X,PAN XB,FENG SL,et al.A experimental investigation about Sibiraea angustata influence upon the ability of antioxidation and immune organs in mice[J].J Lanzhou Univ Med Sci,1994,20(3):143
[13]卫阳飞,刘东花,张宏曦,等.窄叶鲜卑花叶中黄酮和多酚的超声提取工艺及抗氧化性研究[J].中药材,2017,40(1):158WEI YF,LIU DH,ZHANG HX,et al.Ultrasound-assisted extraction technology and antioxidant activity of flavonoids and polyphenols in Sibiraea angustata leaves[J].J Chin Med Mater,2017,40(1):158
[14]陈璐,陶婷婷,王天志.RP-HPLC测定藏药柳茶中阿魏酸和异阿魏酸的含量[J].华西药学杂志,2006,21(2):196CHEN L,TAO TT,WANG TZ.Determination of ferulic acid and isoferulic acid in Sibiraea angustata by RP-HPLC[J].West China J Pharm Sci,2006,21(2):196
[15]闫志慧,陈立书,朱仝飞,等.藏药柳茶叶乙醇水提取物中化学成分的鉴定[J].应用化学,2014,31(2):237YAN ZH,CHEN LS,ZHU TF,et al.Determination of the chemical constituents in aqueous alcohol extract of Sibiraea angustata(Rehd.)Hand-Mazz.leaf[J].Chin J Appl Chem,2014,31(2):237
[16]LI B,CHEN XT,WANG ZW,et al.Two new monoterpenes and one dicaffeic acid ester from Sibiraea angustata with hypolipidemic activities in Hep G2 cells in vitro[J].Phytochem lett,2015,13(7):319