运用在线HPLC-UV-DPPH方法评价地黄药材的抗氧化成分及质量
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摘要
目的:发展在线高效液相色谱-紫外-1,1-二苯基-2-三硝基苯肼(HPLC-UV-DPPH)方法快速筛选地黄药材的抗氧化成分并评价其活性,为中药质量控制提供新的研究思路和模式。方法:运用HPLC-UV-DPPH方法筛选13批地黄药材的抗氧化成分;以毛蕊花糖苷为阳性参比,计算主要抗氧化活性成分的相对活性及总活性;运用高效液相色谱-傅里叶变换质谱(HPLC-FTMS)方法对主要的抗氧化成分进行鉴定;13批地黄样品的总活性结果与《中国药典》方法中毛蕊花糖苷含量的检测结果进行比较分析。结果:HPLC-UV-DPPH方法学考察结果,精密度RSD<4.61%,稳定性RSD<4.25%,重复性RSD<5.28%;建立"量-效"曲线方程为y=0.000 6x4-0.024 1x3+0.404 1x2+1.299x+1.652 1(R2=1);9个抗氧化成分中4个显示较强DPPH抑制峰的成分被鉴定为海胆苷(echinacoside)、焦地黄苯乙醇苷A1/A2(jionoside A1/A2)、毛蕊花糖苷(verbascoside)、异毛蕊花糖苷(isoverbascoside);《中国药典》方法测得毛蕊花糖苷含量较高的4批样品其总活性也较高,而其他样品的结果不呈现正相关,表明除毛蕊花糖苷外的其他成分对总活性的贡献率不应被忽略,质量评价应考虑多成分的测定。结论:研究建立的HPLC-UV-DPPH方法具有良好的重复性和稳定性,化学和生物活性检测相结合为综合评价中药的质量提供了一种新的方法。
OBJECTIVE To develop an on-line HPLC-UV-DPPH method for quick screening of antioxidants and active evaluation of Rehmanniae Radix to provide a new research idea and mode for quality control of traditional Chinese medicine.METHODS HPLC-UV-DPPH was applied to screen antioxidants from 13 batches of Rehmanniae Radix samples.Verbascoside was regarded as a positive reference and used to calculate relative activities and total activities of the main antioxidants.The main antioxidants were identified by HPLC-FTMS.The results of total activities of 13 batches of samples were compared with the detection results of verbascoside content of Chinese Pharmacopoeia method.RESULTS The RSD values of precision,stability and repeatability of HPLC-UV-DPPH method were less than 4.61%,4.25% and 5.28%,respectively.Equation formula of quantity-effect verbascoside was y=0.000 6 x~4+0.024 1 x~3+0.404 1 x~2+1.299 x+1.652 1(R~2= 1).Four antioxidants showing stronger DPPH inhibition peaks in nine antioxidants were identified as echinacoside,jionoside A1/A2,verbascoside,isoverbascoside.Total activities of 4 batches of samples with higher content of verbascoside were higher,while the results of other samples were not positively correlated.This indicated that the contributions of the other components beside of verbascoside should not be ignored.The quality control of Rehmanniae Radix should include the detection of multiple ingredients.CONCLUSION The proposed HPLC-UV-DPPH method is stable and repeatable.The combination of chemical and active detection will provide a new method for quality control of traditional Chinese medicine.
OBJECTIVE To develop an on-line HPLC-UV-DPPH method for quick screening of antioxidants and active evaluation of Rehmanniae Radix to provide a new research idea and mode for quality control of traditional Chinese medicine.METHODS HPLC-UV-DPPH was applied to screen antioxidants from 13 batches of Rehmanniae Radix samples.Verbascoside was regarded as a positive reference and used to calculate relative activities and total activities of the main antioxidants.The main antioxidants were identified by HPLC-FTMS.The results of total activities of 13 batches of samples were compared with the detection results of verbascoside content of Chinese Pharmacopoeia method.RESULTS The RSD values of precision,stability and repeatability of HPLC-UV-DPPH method were less than 4.61%,4.25% and 5.28%,respectively.Equation formula of quantity-effect verbascoside was y=0.000 6 x~4+0.024 1 x~3+0.404 1 x~2+1.299 x+1.652 1(R~2= 1).Four antioxidants showing stronger DPPH inhibition peaks in nine antioxidants were identified as echinacoside,jionoside A1/A2,verbascoside,isoverbascoside.Total activities of 4 batches of samples with higher content of verbascoside were higher,while the results of other samples were not positively correlated.This indicated that the contributions of the other components beside of verbascoside should not be ignored.The quality control of Rehmanniae Radix should include the detection of multiple ingredients.CONCLUSION The proposed HPLC-UV-DPPH method is stable and repeatable.The combination of chemical and active detection will provide a new method for quality control of traditional Chinese medicine.
引文
[1]Dimitrios B.Sources of natural phenolic antioxidants[J].Trends Food Sci Technol,2006,17(9):505-512.
[2]Mirzaei B,Rahmani-Nia F,Salehi Z,et al.Effects of creatine monohydrate supplementation on oxidative DNA damage and lipid peroxidation induced by acute incremental exercise to exhaustion in wrestlers[J].Kinesiology,2013,45(1):30-40.
[3]Kumar S,Sharma UK,Sharma AK,et al.Protective efficacy of Solanum xanthocarpum root extracts against free radical damage:phytochemical analysis and antioxidant effect[J].Cell Mol Biol(Noisy-le-grand),2012,58(1):174-181.
[4]Sun LQ,Ding XP,Qi J,et al.Antioxidant anthocyanins screening through spectrum-effect relationships and DPPHHPLC-DAD analysis on nine cultivars of introduced rabbiteye blueberry in China[J].Food Chem,2012,132(2):759-765.
[5]Li F,Zhang LD,Li BC,et al.Screening of free radical scavengers from Erigeron breviscapus using on-line HPLC-ABTS/DPPH based assay and mass spectrometer detection[J].Free Radic Res,2012,46(3):286-294.
[6]Wang G,Huang X,Pei D,et al.DPPH-HPLC-DAD analysis combined HSCCC for screening and identification of radical scavengers in Cynomorium songaricum Rupr[J].New J Chem,2016,40(4):3885-3891.
[7]Tong S,Chu C,Wei Y,et al.Preparation and effects of 2,3-dehydrosilymarin,apromising and potent antioxidant and free radical scavenger[J].J Pharm Pharmacol,2011,63(2):238-244.
[8]Geng DD,Dong Q,Tan L,et al.On-line screening and identification of free radical scavenging compounds in Salvia miltiorrhiza Bunge and Salvia prattiiaHemsl.by HPLC-DAD-ESI/MSn-DPPH[J].J Instrum Anal(分析测试学报),2015,34(3):314-320.
[9]Feng WS,Li M,Zheng XK,et al.Study on chemical constituents of immunosuppressive parts from the Roots of Rehmannia glutinosa[J].Chin Pharm J(中国药学杂志),2014,49(17):1496-1502.
[10]Liu YF,Liang D,Luo H,et al.Chemical constituents from root tubers of Rehmannia glutinosa[J].Chin Tradit Herb Drugs(中草药),2014,45(1):16-22.
[11]Tao Y,Jiang YH,Tang KJ,et al.Comparison of chemical constituents of crude and processed products of Rehmannia glutinosa by using UHPLC-Q-TOF/MS[J].Tradit Chin Drug Res Pharmacol(中药新药与临床药理),2016,1:102-106.
[12]Li HF.Summary on pharmacological action and clinical usage of Rehmanniae Radix[J].J Pharm Res(药学研究),2014,6:345-347.
[13]Li HW,Meng XL.Research progress on chemical constituents and pharmacological activities of Rehmannia glutinosa[J].Drug Eval Res(药物评价研究),2015,38(2):218-228.
[14]Ch.P(2015)Vol I(中国药典2015年版一部)[S].2015:124-125.
[15]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.
[16]Song QQ,Zhao YF,Zhang N,et al.Establishment of HPLC fingerprint of Rehmanniae Radix and its HPLC-ESI-MS analysis[J].Chin Tradit Herb Drugs(中草药),2016,47(23):4247-4252.
[17]Zhang N,Song Y,Song Q,et al.Qualitative and quantitative assessments of Aconiti Lateralis Radixpraeparata using highperformance liquid chromatography coupled with diode array detection and hybrid ion trap-time-of-flight mass spectrometry[J].J Chromatogr Sci,2016,54(6):888-901.
[18]Zhang BY,Jiang ZZ,Wang RF,et al.Analysis of chemical constituents in fresh,dried and prepared Rehmanniae Radix by UPLC/ESI-Q-TOF M[J].Chin Tradit Pat Med(中成药),2016,38(5):1104-1108.
[2]Mirzaei B,Rahmani-Nia F,Salehi Z,et al.Effects of creatine monohydrate supplementation on oxidative DNA damage and lipid peroxidation induced by acute incremental exercise to exhaustion in wrestlers[J].Kinesiology,2013,45(1):30-40.
[3]Kumar S,Sharma UK,Sharma AK,et al.Protective efficacy of Solanum xanthocarpum root extracts against free radical damage:phytochemical analysis and antioxidant effect[J].Cell Mol Biol(Noisy-le-grand),2012,58(1):174-181.
[4]Sun LQ,Ding XP,Qi J,et al.Antioxidant anthocyanins screening through spectrum-effect relationships and DPPHHPLC-DAD analysis on nine cultivars of introduced rabbiteye blueberry in China[J].Food Chem,2012,132(2):759-765.
[5]Li F,Zhang LD,Li BC,et al.Screening of free radical scavengers from Erigeron breviscapus using on-line HPLC-ABTS/DPPH based assay and mass spectrometer detection[J].Free Radic Res,2012,46(3):286-294.
[6]Wang G,Huang X,Pei D,et al.DPPH-HPLC-DAD analysis combined HSCCC for screening and identification of radical scavengers in Cynomorium songaricum Rupr[J].New J Chem,2016,40(4):3885-3891.
[7]Tong S,Chu C,Wei Y,et al.Preparation and effects of 2,3-dehydrosilymarin,apromising and potent antioxidant and free radical scavenger[J].J Pharm Pharmacol,2011,63(2):238-244.
[8]Geng DD,Dong Q,Tan L,et al.On-line screening and identification of free radical scavenging compounds in Salvia miltiorrhiza Bunge and Salvia prattiiaHemsl.by HPLC-DAD-ESI/MSn-DPPH[J].J Instrum Anal(分析测试学报),2015,34(3):314-320.
[9]Feng WS,Li M,Zheng XK,et al.Study on chemical constituents of immunosuppressive parts from the Roots of Rehmannia glutinosa[J].Chin Pharm J(中国药学杂志),2014,49(17):1496-1502.
[10]Liu YF,Liang D,Luo H,et al.Chemical constituents from root tubers of Rehmannia glutinosa[J].Chin Tradit Herb Drugs(中草药),2014,45(1):16-22.
[11]Tao Y,Jiang YH,Tang KJ,et al.Comparison of chemical constituents of crude and processed products of Rehmannia glutinosa by using UHPLC-Q-TOF/MS[J].Tradit Chin Drug Res Pharmacol(中药新药与临床药理),2016,1:102-106.
[12]Li HF.Summary on pharmacological action and clinical usage of Rehmanniae Radix[J].J Pharm Res(药学研究),2014,6:345-347.
[13]Li HW,Meng XL.Research progress on chemical constituents and pharmacological activities of Rehmannia glutinosa[J].Drug Eval Res(药物评价研究),2015,38(2):218-228.
[14]Ch.P(2015)Vol I(中国药典2015年版一部)[S].2015:124-125.
[15]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.
[16]Song QQ,Zhao YF,Zhang N,et al.Establishment of HPLC fingerprint of Rehmanniae Radix and its HPLC-ESI-MS analysis[J].Chin Tradit Herb Drugs(中草药),2016,47(23):4247-4252.
[17]Zhang N,Song Y,Song Q,et al.Qualitative and quantitative assessments of Aconiti Lateralis Radixpraeparata using highperformance liquid chromatography coupled with diode array detection and hybrid ion trap-time-of-flight mass spectrometry[J].J Chromatogr Sci,2016,54(6):888-901.
[18]Zhang BY,Jiang ZZ,Wang RF,et al.Analysis of chemical constituents in fresh,dried and prepared Rehmanniae Radix by UPLC/ESI-Q-TOF M[J].Chin Tradit Pat Med(中成药),2016,38(5):1104-1108.