基于HPLC-UV-DPPH法的地黄和熟地黄药材抗氧化活性成分比较研究
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摘要
目的建立HPLC-UV-DPPH在线分析方法,比较不同生产企业的地黄药材在炮制前后抗氧化活性成分的变化,为中药炮制及质量控制提供有效的评价方法。方法 HPLC-UV-DPPH在线联用筛选系统中HPLC流动相为乙腈-0.1%醋酸水溶液,梯度洗脱,体积流量为1.0 m L/min,色谱柱为Aglient Extend C18柱(250 mm×4.6 mm,5μm),柱温30℃,检测波长334 nm,DPPH溶液体积流量0.5 m L/min,检测波长517 nm。以毛蕊花糖苷为阳性对照,通过"量-效"研究思路评价样品的总抗氧化活性及各成分对总抗氧化活性的贡献率。采用HPLC-FT-MS对药材中主要的抗氧化活性成分进行鉴定并比较药材炮制前后的差异。结果 HPLC-UV-DPPH方法检测到地黄中主要含有9个抗氧化活性成分,而熟地黄含有13个,地黄和熟地黄有8个共有抗氧化活性成分,地黄在炮制前后抗氧化活性成分明显不同,不同生产企业的样品中各抗氧化活性成分的活性差异较大。抗氧化活性贡献率较高的成分分别为玉叶金花苷酸、海胆苷、焦地黄苯乙醇苷A1/A2、毛蕊花糖苷、异毛蕊花糖苷。结论 HPLC-UV-DPPH法稳定、灵敏、重现性好,适用于地黄和熟地黄中抗氧化活性成分的快速筛选和质量评价。
Objective To develop an HPLC-UV-DPPH method to compare anti-oxidants of Rehmanniae Radix and Rehmanniae RadixPraeparata sample from different manufactories and to provide an effective method for the processing and quality control of traditional Chinese medicine. Methods HPLC in HPLC-UV-DPPH system was performed on Aglient Extend C18(250 mm × 4.6 mm, 5 μm) column with gradient elution of acetonitrile-0.1% acetic acid at the flow rate of 1.0 m L/min, and the detection wavelength was at 334 nm. The column temperature was 30 ℃. Flow rate of DPPH solution is 0.5 m L/min, and detection wavelength was set at 517 nm. The total activities of the samples and the contribution rate of each component to the total activity were evaluated by the "quantity-effect" research idea, and verbascoside was regarded as a positive reference. The main anti-oxidants in original and processed herbs were identified by HPLC-FT-MS and were compared. Results The detection of HPLC-UV-DPPH method showed that there were nine anti-oxidants in Rehmanniae Radix extract, while 13 anti-oxidants were found in Rehmanniae Radix Praeparata. There were eight common anti-oxidants in the two herbs. The anti-oxidants were obviously different before and after Rehmanniae Radix processed. The activities of the antioxidants in different samples were markedly different. Anti-oxidants with higher contributions were mussaenosidic acid, echinacoside, jionoside A1/A2, verbascoside, and isoverbascoside, respectively. Conclusion The HPLC-UV-DPPH method is stable, sensitive, reproducible, and suitable for rapid screening of anti-oxidants and quality evaluation of Rehmanniae Radix and Rehmannia Radix Praeparata.
Objective To develop an HPLC-UV-DPPH method to compare anti-oxidants of Rehmanniae Radix and Rehmanniae RadixPraeparata sample from different manufactories and to provide an effective method for the processing and quality control of traditional Chinese medicine. Methods HPLC in HPLC-UV-DPPH system was performed on Aglient Extend C18(250 mm × 4.6 mm, 5 μm) column with gradient elution of acetonitrile-0.1% acetic acid at the flow rate of 1.0 m L/min, and the detection wavelength was at 334 nm. The column temperature was 30 ℃. Flow rate of DPPH solution is 0.5 m L/min, and detection wavelength was set at 517 nm. The total activities of the samples and the contribution rate of each component to the total activity were evaluated by the "quantity-effect" research idea, and verbascoside was regarded as a positive reference. The main anti-oxidants in original and processed herbs were identified by HPLC-FT-MS and were compared. Results The detection of HPLC-UV-DPPH method showed that there were nine anti-oxidants in Rehmanniae Radix extract, while 13 anti-oxidants were found in Rehmanniae Radix Praeparata. There were eight common anti-oxidants in the two herbs. The anti-oxidants were obviously different before and after Rehmanniae Radix processed. The activities of the antioxidants in different samples were markedly different. Anti-oxidants with higher contributions were mussaenosidic acid, echinacoside, jionoside A1/A2, verbascoside, and isoverbascoside, respectively. Conclusion The HPLC-UV-DPPH method is stable, sensitive, reproducible, and suitable for rapid screening of anti-oxidants and quality evaluation of Rehmanniae Radix and Rehmannia Radix Praeparata.
引文
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[23]耿丹丹,董琦,谭亮,等.HPLC-DAD-ESI/MSnDPPH在线筛选与鉴别丹参和康定鼠尾草中抗氧化活性成分[J].分析测试学报,2015,34(3):314-320.
[24]宋子荣,谭梓骏,陈西松,等.地黄提取工艺的优化[J].中国实验方剂学杂志,2006,12(1):8-9.
[25]Peng W,Zeng Q,Li D,et al.Multiple on-line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection[J].Biomed Chromatogr,2016,30(11):1854-1860.
[26]宋青青,赵云芳,张娜,等.生地黄HPLC指纹图谱的建立及其HPLC-ESI-MS分析[J].中草药,2016,47(23):4247-4252.
[2]王虹,陈军辉,赵恒强,等.高效液相色谱-质谱-二苯基三硝基苯肼在线筛选与鉴别茶叶中抗氧化活性成分[J].分析化学,2009,37(6):795-800.
[3]Raudonis R,Jakstas V,Burdulis D,et al.Investigation of contribution of individual constituents to antioxidant activity in herbal drugs using postcolumn HPLC method[J].Medicina,2009,45(5):382-394.
[4]Li S Y,Yu Y,Li S P.Identification of antioxidants in essential oil of Radix Angelicae Sinensis using HPLC coupled with DAD-MS and ABTS-based assay[J].JAgric Food Chem,2007,55(9):3358-3362.
[5]Dapkevicius A,van Beek T A,Niederl?nder H A.Evaluation and comparison of two improved techniques for the on-line detection of antioxidants in liquid chromatography eluates[J].J Chromatogr A,2001,912(1):73-82.
[6]朱卉,朱丹妮,余伯阳.HPLC-CL联用在线检测消癌平注射液清除H2O2及O·2自由基活性[J].中成药,2010,32(8):1386-1390.
[7]Wu L,Ding X P,Zhu D N,et al.Study on the radical scavengers in the traditional Chinese medicine formula shengmai san by HPLC-DAD coupled with chemiluminescence(CL)and ESI-MS/MS[J].J PharmBiomed Anal,2010,52(4):438-445.
[8]Ding X P,Zhang C L,Qi J,et al.The Spectrum-effect integrated fingerprint of Polygonum cuspidatum based on HPLC-diode array detection-flow injectionchemiluminescence[J].Chin J Nat Med,2013,11(5):546-552.
[9]Kara?elik A A,Kü?ük M,?skefiyeli Z,et al.Antioxidant components of Viburnum opulus L.determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESIMS methods[J].Food Chem,2015,175(5):106-114.
[10]He L,Zhang X,Xu H,et al.Subcritical water extraction of phenolic compounds from pomegranate(Punica granatum L.)seed residues and investigation into their antioxidant activities with HPLC-ABTS+assay[J].Food Bioprod Process,2012,90(2):215-223.
[11]张雷,丁晓萍,戚进,等.HPLC-DPPH在线联用技术评价茶的抗氧化活性[J].中国药科大学学报,2012,43(3):236-240.
[12]Sun L Q,Ding X P,Qi J,et al.Antioxidant anthocyanins screening through spectrum-effect relationships and DPPH-HPLC-DAD analysis on nine cultivars of introduced rabbiteye blueberry in China[J].Food Chem,2012,132(2):759-765.
[13]?elik S E,?zyürek M,Gü?lüK,et al.Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity(CUPRAC)assay with post-column detection[J].Anal Chim Acta,2010,674(1):79-88.
[14]Le G J,Guffond D,Hamon E,et al.Combined microplate-ABTS and HPLC-ABTS analysis of tomato and pepper extracts reveals synergetic and antagonist effects of their lipophilic antioxidative components[J].Food Chem,2017,223(5):62-71.
[15]中国药典[S].一部.2015.
[16]高森,李正翔,赵鹏,等.高效液相色谱指纹图谱在熟地黄质量稳定性中的应用研究[J].中国医院用药评价与分析,2013,13(10):908-910.
[17]曾艳,贾正平,张汝学.地黄化学成分及药理研究进展[J].中成药,2006,28(4):609-611.
[18]李鹏飞,苗明三.熟地黄的现代研究及应用现状分析[J].中医学报,2014,29(2):252-254.
[19]李建军,李静云,王莹,等.地黄药用研究概述[J].生物学教学,2013,38(3):4-7.
[20]李红伟,孟祥乐.地黄化学成分及其药理作用研究进展[J].药物评价研究,2015,38(2):218-228.
[21]王晓飞,焦海胜,李玉民.HPLC-柱后生物活性检测在线联用技术在天然活性成分筛选中的应用[J].中草药,2013,44(8):1047-1051.
[22]Bandonien D,Murkovic M.The detection of radical scavenging compounds in crude extract of borage(Borago officinalis L.)by using an on-line HPLC-DPPH method[J].J Biochem Biophys Methods,2002,53(1/3):45-49.
[23]耿丹丹,董琦,谭亮,等.HPLC-DAD-ESI/MSnDPPH在线筛选与鉴别丹参和康定鼠尾草中抗氧化活性成分[J].分析测试学报,2015,34(3):314-320.
[24]宋子荣,谭梓骏,陈西松,等.地黄提取工艺的优化[J].中国实验方剂学杂志,2006,12(1):8-9.
[25]Peng W,Zeng Q,Li D,et al.Multiple on-line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection[J].Biomed Chromatogr,2016,30(11):1854-1860.
[26]宋青青,赵云芳,张娜,等.生地黄HPLC指纹图谱的建立及其HPLC-ESI-MS分析[J].中草药,2016,47(23):4247-4252.