知母及其不同炮制品的指纹图谱建立与抗氧化活性的谱-效关系研究
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篇名: 知母及其不同炮制品的指纹图谱建立与抗氧化活性的谱-效关系研究
TITLE: Establishment of Fingerprint and Spectrum-effect Relationship Study on Anti-oxidantion Activity of Anemarrhena asphodeloides and Its Different Processed Products
摘要: 目的:建立知母及其不同炮制品乙醇提取物和丙酮提取物的指纹图谱,并考察其与抗氧化活性的谱-效关系。方法:分别采用高效液相色谱法(HPLC)和高效液相色谱-蒸发光散射检测法(HPLC-ELSD),色谱条件分别为色谱柱ThermoBDSHypersilC18、流动相乙腈-0.2%醋酸水溶液、流速1.0mL/min、柱温30℃、检测波长258nm、进样量10μL以及色谱柱XDB-C18、流动相乙腈-0.2%醋酸水溶液(梯度洗脱)、流速0.9mL/min、柱温30℃、雾化器温度40℃、氮气流速1.6mL/min、进样量10μL。分别以芒果苷、知母皂苷BⅡ为参照,采用《中药色谱指纹图谱相似度评价系统(2004A版)》绘制20批知母及其炮制品乙醇提取物和丙酮提取物的指纹图谱并进行相似度评价,标定共有峰。以1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率为指标,考察20批知母及其炮制品乙醇提取物和丙酮提取物的抗氧化活性。以DPPH自由基清除率为因变量、共有峰峰面积为自变量,采用偏最小二乘回归法分别分析知母乙醇提取物和丙酮提取物与抗氧化活性的谱-效关系。结果:从20批知母炮制品乙醇提取物指纹图谱中标定出共有峰8个(M1~M8),指认出芒果苷(色谱峰M7);相似度为0.389~1.000;从丙酮提取物指纹图谱中标定出共有峰7个(S1~S7),指认出知母皂苷BⅡ(色谱峰S5);相似度为0.044~0.999。20批知母及其炮制品乙醇提取物的DPPH自由基清除率为21.23%~81.39%,生知母显著低于盐知母、酒知母(P<0.001);丙酮提取物的DPPH自由基清除率为49.73%~83.78%,生知母显著高于盐知母、酒知母、炒知母(P<0.001)。知母乙醇提取物图谱中峰M2~M7的标准化回归系数均大于0,与抗氧化活性成正相关;仅峰M7的变量重要性投影(VIP)值大于1。知母丙酮提取物图谱中峰S4~S7的标准化回归系数均大于0,与抗氧化活性成正相关;VIP值大小排序依次为峰S5>S6>S4,且VIP值均大于1。结论:成功建立知母乙醇提取物和丙酮提取物的指纹图谱。芒果苷(峰M7)可能是知母乙醇提取物抗氧化作用的主要药效物质;知母皂苷BⅡ(峰S5)、峰S6、峰S4可能是知母丙酮提取物抗氧化作用的主要药效物质。
ABSTRACT: OBJECTIVE:To establish the fingerprint of ethanol extract and acetone extract from Anemarrhena asphodeloides and its different processed products ,and to investigate the spectrum-effect relationship between the fingerprint and the antioxidant activity. METHODS :HPLC method and HPLC-ELSD method were adopted. The determination was performed on Thermo BDS Hypersil C 18 column with mobile phase consisted of acetonitrile- 0.2% acetic acid at the flow rate of 1.0 mL/min. The column temperature was 30 ℃,and the detection wavelength was set at 258 nm. The sample size was 10 μL. The determination was performed on XDB-C 18 columnwith mobile phase consisted of acetonitrile-0.1% acetic acid (gradient elution )at the flow rate of 0.9 mL/min. The column temperature was 30 ℃ . The temperature of atomizer was 40 ℃ and the flow rare of N 2 was 1.6 mL/min. The sample size was 10 μL. Using mangiferin and timosaponin B Ⅱ as reference ,Fingerprint Similarity Eva- com luation System of TCM Chromatogram (2004A edition )was adopted to draw the fingerprint of ethanol extract and acetoneextract from 20 batches of A. asphodeloides and its different processed products to confirm common peaks. Using scave nging rate of 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)radical as index,antioxidant activities of ethanol extract and acetone extract from 20 batches of A. asphodeloides and its processed products were investigated. Using scavenging rate of DPPH radical as dependent variable ,common peak area as independent variable ,PLSR was used to analyze the spectrum-effect relationship of ethanol extract and acetone extract from A. asphodeloides with antioxidantion activity. RESULTS :Eight peaks (M1-M8)were identified in the fingerprints of ethanol extracts from 20 batches of processed A. asphodeloides . Mangiferin (chromatogram peak M 7)was identified with similarity of 0.389-1.000;seven comon peaks (S1-S7)and timosaponin B Ⅱ(peak S 5)were identified in the fingerprint of acetone extract ,and the similarity was 0.044-0.999. DPPH radical scavenging rate of ethanol extract from 20 batches of A. asphodeloides and its processed products was 21.23%- 81.39%,and A. asphodeloides was significantly lower than salt-processed A. asphodeloides with salt wine-processed A. asphodeloides (P<0.001);and that of acetone extract was 49.73%-83.78%,and A. asphodeloides was significantly higher than stir-baked A. asphodeloides with salt ,wine or fire (P<0.001). The standardized regression coefficients of peaks M 2-M7 in the spectrum of ethanol extract from A. asphodeloides were all greater than 0,which was positively correlated with antioxidant activity. Only the variable importance projection (VIP)value of peak M 7 was greater than 1,which had an important contribution. The standardized regression coefficients of peaks S 4-S7 in the acetone extract spectrum of A. asphodeloides were greater than 0,and were positively correlated with antioxidant activity. The order of VIP values was peak S 5>S6>S4,and the VIP values were all greater than 1. CONCLUSIONS:The fingerprint of the different processed products A. asphodeloides and its antioxidant activity spectral effect relationship were successfully established ;mangiferin(peak M 7)may be the main antioxidant substance of ethanol extract from A. asphodeloides . Timosaponin B Ⅱ(peak S 5),peak S 6 and peak S 4 may be the main antioxidant substance in acetone extract from A. asphodeloides .
期刊: 2020年第31卷第22期
作者: 贾毓欣,刘海帆,司明东,李新蕊,宋军娜,郑玉光,马东来
AUTHORS: JIA Yuxin,LIU Haifan ,SI Mingdong ,LI Xinrui ,SONG Junna,ZHENG Yuguang ,MA Donglai
关键字: 知母;乙醇提取物;丙酮提取物;抗氧化活性;谱-效关系;偏最小二乘回归法
KEYWORDS: Anemarrhena asphodeloides;Ethanol extract ;Acetone extract ;Antioxidant acitivity ;Spectrum-effect relationship ;
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