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Volume 42 Issue 5
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LAI Liyong, XIA Tianshuang, XU Shengyan, JIANG Yiping, YUE Xiaoqiang, XIN Hailiang. Study on spectrum-effect relationship based on antioxidant activity of Artemisiae Annuae Herba[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 203-210, 216. doi: 10.12206/j.issn.2097-2024.202211012
Citation: LAI Liyong, XIA Tianshuang, XU Shengyan, JIANG Yiping, YUE Xiaoqiang, XIN Hailiang. Study on spectrum-effect relationship based on antioxidant activity of Artemisiae Annuae Herba[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 203-210, 216. doi: 10.12206/j.issn.2097-2024.202211012

Study on spectrum-effect relationship based on antioxidant activity of Artemisiae Annuae Herba

doi: 10.12206/j.issn.2097-2024.202211012
  • Received Date: 2022-11-07
  • Rev Recd Date: 2024-01-26
  • Available Online: 2024-05-22
  • Publish Date: 2024-05-25
  •   Objective  To screen the pharmacodynamic material basic components of Artemisiae Annuae Herba and study its antioxidant activity in vitro by investigating the spectrum-effect relationship between the HPLC fingerprints of 11 batches of Artemisiae Annuae Herba (dried aerial part of Artemisia annua L.).   Methods   The determination was performed on Aglient C18 column (250 mm×4.6 mm, 5 μm) with mobile phase consisted of 0.2% phosphoric acid solution-Methanol (gradient elution) at the flow rate of 1.0 ml/min. The column temperature was indoor temperature, and detection wavelength was 220 nm, with sample size of 10 μl. Using isochlorogenic acid A as reference, HPLC fingerprints of 11 batches of samples were determined. The common peaks of 11 batches of samples were identified and recorded through TCM chromatographic fingerprint similarity evaluation system (2012 edition). Using scavenging rate of DPPH and ABTS free radical as pharmacodynamic indicators of antioxidant effects, SIMCA 14.1 analysis software was used for PLSR to establish the spectra-effect relationship.   Results   There were 48 common peaks on 11 batches of sample, 11 components were identified as scopoletin, scoparone, isochlorogenic acid B, A, C, luteolin, apigenin, chrysosplenetin, artemisinin, artemisetin and artemisinic acid. The scavenging activity of 11 batches of samples to DPPH and ABTS free radicals was detected. The spectrum-effect relationship showed that isochlorogenic acid A, B, C and scoparone were positively associated with its antioxidant capacity, and variable projection value was greater than 1. It was suggested that these components were the material basis of antioxidant effect in Artemisiae Annuae Herba.   Conclusion   This study investigates the antioxidant capacity of different substances in Artemisiae Annuae Herba in vitro, and proves that isochlorogenic acid A,B, C and scoparone play a major role for the antioxidant capacity.
  • [1] 张兴. 认识身边的中药: 青蒿[J]. 中医健康养生, 2021, 7(4):26-27.
    [2] 徐明霞. 青蒿素及其衍生物在动物生产中的应用研究[J]. 饲料研究, 2021, 44(4):147-149.
    [3] SKOWYRA M, GALLEGO M G, SEGOVIA F, et al. Antioxidant properties of Artemisia annua extracts in model food emulsions[J]. Antioxidants (Basel), 2014, 3(1):116-128. doi:  10.3390/antiox3010116
    [4] 赖立勇, 徐圣焱, 夏天爽, 等. 基于抗氧化机制的中药及其化学成分在骨质疏松中的应用[J]. 海军军医大学学报, 2022(8):943-950.
    [5] 国家药典委员会. 中华人民共和国药典-一部: 2020年版[M]. 北京: 中国医药科技出版社, 2020.
    [6] 张秋红, 朱子微, 李晋, 等. 中药青蒿化学成分与种植研究现状[J]. 中国医药导报, 2011, 8(19):10-12. doi:  10.3969/j.issn.1673-7210.2011.19.004
    [7] WANG Q, XIAO L. Isochlorogenic acid A attenuates acute lung injury induced by LPS via Nf-κB/NLRP3 signaling pathway[J]. Am J Transl Res, 2019, 11(11):7018-7026.
    [8] SROKA Z, CISOWKI W. Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids[J]. Food Chem Toxicol, 2003, 41(6):753-758. doi:  10.1016/S0278-6915(02)00329-0
    [9] WITAICENIS A, SEITO L N, DA SILVEIRA CHAGAS A, et al. Antioxidant and intestinal anti-inflammatory effects of plant-derived coumarin derivatives[J]. Phytomedicine, 2014, 21(3):240-246. doi:  10.1016/j.phymed.2013.09.001
    [10] 刘晓燕, 蒋益萍, 张嘉宝等. 啤酒花的HPLC指纹图谱建立及其抗氧化作用谱效关系研究[J]. 中国药房, 2020, 31(02):138-143. doi:  10.6039/j.issn.1001-0408.2020.02.03
    [11] 朱亚莹, 吴啟南, 段慧芳等. 青蒿UPLC指纹图谱的建立与多种成分含量测定[J]. 中药材, 2019, 42(10):2323-2329.
    [12] YAN F X, WANG H T, GAO Y, et al. Artemisinin protects retinal neuronal cells against oxidative stress and restores rat retinal physiological function from light exposed damage[J]. ACS Chem Neurosci, 2017, 8(8):1713-1723. doi:  10.1021/acschemneuro.7b00021
    [13] EFFERTH T. From ancient herb to modern drug: Artemisia annua and artemisinin for cancer therapy[J]. Semin Cancer Biol, 2017, 46:65-83. doi:  10.1016/j.semcancer.2017.02.009
    [14] JONE D P. Radical-free biology of oxidative stress[J]. Am J Physiol Cell Physiol, 2008, 295(4):C849-68. doi:  10.1152/ajpcell.00283.2008
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Study on spectrum-effect relationship based on antioxidant activity of Artemisiae Annuae Herba

doi: 10.12206/j.issn.2097-2024.202211012

Abstract:   Objective  To screen the pharmacodynamic material basic components of Artemisiae Annuae Herba and study its antioxidant activity in vitro by investigating the spectrum-effect relationship between the HPLC fingerprints of 11 batches of Artemisiae Annuae Herba (dried aerial part of Artemisia annua L.).   Methods   The determination was performed on Aglient C18 column (250 mm×4.6 mm, 5 μm) with mobile phase consisted of 0.2% phosphoric acid solution-Methanol (gradient elution) at the flow rate of 1.0 ml/min. The column temperature was indoor temperature, and detection wavelength was 220 nm, with sample size of 10 μl. Using isochlorogenic acid A as reference, HPLC fingerprints of 11 batches of samples were determined. The common peaks of 11 batches of samples were identified and recorded through TCM chromatographic fingerprint similarity evaluation system (2012 edition). Using scavenging rate of DPPH and ABTS free radical as pharmacodynamic indicators of antioxidant effects, SIMCA 14.1 analysis software was used for PLSR to establish the spectra-effect relationship.   Results   There were 48 common peaks on 11 batches of sample, 11 components were identified as scopoletin, scoparone, isochlorogenic acid B, A, C, luteolin, apigenin, chrysosplenetin, artemisinin, artemisetin and artemisinic acid. The scavenging activity of 11 batches of samples to DPPH and ABTS free radicals was detected. The spectrum-effect relationship showed that isochlorogenic acid A, B, C and scoparone were positively associated with its antioxidant capacity, and variable projection value was greater than 1. It was suggested that these components were the material basis of antioxidant effect in Artemisiae Annuae Herba.   Conclusion   This study investigates the antioxidant capacity of different substances in Artemisiae Annuae Herba in vitro, and proves that isochlorogenic acid A,B, C and scoparone play a major role for the antioxidant capacity.

LAI Liyong, XIA Tianshuang, XU Shengyan, JIANG Yiping, YUE Xiaoqiang, XIN Hailiang. Study on spectrum-effect relationship based on antioxidant activity of Artemisiae Annuae Herba[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 203-210, 216. doi: 10.12206/j.issn.2097-2024.202211012
Citation: LAI Liyong, XIA Tianshuang, XU Shengyan, JIANG Yiping, YUE Xiaoqiang, XIN Hailiang. Study on spectrum-effect relationship based on antioxidant activity of Artemisiae Annuae Herba[J]. Journal of Pharmaceutical Practice and Service, 2024, 42(5): 203-210, 216. doi: 10.12206/j.issn.2097-2024.202211012
  • 中药青蒿为植物菊科黄花蒿(Artemisia annua L.)的干燥地上部分,味苦、辛,性寒,归肝、胆经,具有清虚热等功效[1]。现代研究表明,青蒿及其所含有关单体成分除了在抗疟疾方面有效外,在抑菌抗炎、免疫调节和缓解热应激等方面也有效果[2],特别是在抗氧化方面,青蒿亦表现出良好作用[3]。现有研究表明,氧化应激与许多疾病关系密切,过度氧化应激可导致骨质疏松症等疾病[4]。目前,对青蒿所含活性成分青蒿素研究较多,特别是其抗疟、抗氧化等作用受到关注[5],但青蒿素之外的其他成分在抗氧化方面亦有活性[6-9],应受到重视。本研究进行青蒿抗氧化活性的谱效关系研究,分析色谱特征峰与DPPH和ABTS自由基清除能力的关联性[10],探究青蒿所含不同化学成分的抗氧化活性,以阐明青蒿体外抗氧化活性的药效物质基础,为青蒿的质量评价和资源开发等提供依据。

    • 岛津LC-20AD型高效液相色谱仪,包括四元泵、PAD检测器、自动进样器(日本岛津株式会社);BT285S型电子分析天平(德国Sartorius 公司);DL-1000B型超声波清洗仪(上海之信仪器有限公司);ELx 800型多功能酶标仪(美国 Biotek公司)。

    • 东莨菪内酯(批号:20220215)、芹菜素(批号:20220216)、木犀草素(批号:20220216)购自上海历鼎生物技术有限公司;异绿原酸A(批号:DSTDY054701)、异绿原酸B(批号:DST210625-037)、异绿原酸C(批号:DSTDY003802)、滨蒿内酯(批号:DSTDB003101)、艾黄素(批号:DST220119-004)、青蒿素(批号:DSTDQ004702)、青蒿酸(批号:DST210919-048)购自成都德思特生物技术有限公司;所有对照品纯度均≥98%。DPPH自由基清除能力试剂盒(批号:20220223)、ABTS自由基清除能力试剂盒(批号:20220224)购自上海励瑞生物科技有限公司。

    • 11批青蒿药材样品信息见表1,经海军军医大学药学系生药教研室辛海量教授鉴定为Artemisia annua L.的干燥地上部分。

      序号 产地 序号 产地
      S1 河北邯郸 S7 安徽亳州
      S2 安徽亳州 S8 江西赣州
      S3 河南驻马店 S9 四川自贡
      S4 安徽亳州 S10 河南南阳
      S5 湖北恩施 S11 重庆市
      S6 安徽亳州
    • 采用 Agilent C18(250 mm×4.6 mm, 5 μm)色谱柱;流动相为0.2%磷酸水溶液(A)-甲醇(B),梯度洗脱(0~5 min,90%→82.5% A;5~10 min,82.5%→75% A;10~30 min,75%→62% A;30~38 min,62%→58.3% A;38~43 min,58.3% A;43~48 min,58.3%→55% A;48~58 min,55→40% A;58~68 min,40%→20% A;68~72 min,20%→18.5% A;72~80 min,18.5%→5% A;80~90min,5%→90% A);流速:1 ml /min;柱温:室温;检测波长:220 nm;进样量:10 μl。

    • 精密称取青蒿素、艾黄素、青蒿酸、猫眼草黄素、芹菜素、木犀草素、异绿原酸C、异绿原酸A、异绿原酸B、滨蒿内酯、东莨菪内酯对照品适量,用甲醇溶解得到浓度分别为300、31、96、78 、16、12、52、134、28、18、86 μg/ml的混合对照品溶液[11]

    • 药材粉末过50目筛后,精密称取1.5 g,加入甲醇50 ml,记录重量后超声(600 W,40 kHz,50 ℃)提取50 min,取出放置冷却至室温后加甲醇至之前的重量,混匀,过0. 45 μm微孔滤膜。

    • 取“2.2.2”项下编号为S10的供试品溶液适量,按“2.1”项所述条件连续运行6次,计算得到RSD(色谱峰保留时间)均小于0.49%,RSD(峰面积)均小于1.93%,表明本研究所用设备精密度良好。

    • 取“2.2.2”项下编号为S10的供试品溶液适量,分别于室温下放置0、2、4、8、12、16、24 h时按“2.1”项所述条件运行,计算得到RSD(色谱峰保留时间)均小于0.69%,RSD(峰面积)均小于4.67%,说明配制的溶液在1 d内具有稳定性。

    • 取编号为S10的青蒿样品粉末1.5 g,共6份,精密称定,按“2.2.2”项和“2.1”项下制备供试品溶液并运行,计算得到色谱峰保留时间均小于0.72%,峰面积均小于4.76%,表明该方法重复性良好。

    • 分别取11批青蒿样品,按照“2.2.2”项下方法制备供试品溶液,精密吸取供试品溶液10 μl,按“2.1”项下色谱条件进样检测后,将获得的HPLC数据导入《中药色谱指纹图谱相似度评价系统》(2012版),得到11批青蒿样品的指纹图谱,指认出48个共有峰,见图1图2

    • 11批青蒿样品共有48个共有峰,共有峰占总峰比在0.776~0.864之间。选择S10为参照图谱,时间窗宽度设置为0.1 min,采用平均数法,以指纹图谱中峰形较好的色谱峰进行多点校正后生成叠加图谱,即为对照图谱。通过与混合对照品的HPLC分析结果对比,指认出9号峰为东莨菪内酯、14号峰为滨蒿内酯、15号峰为异绿原酸B、16号峰为异绿原酸A、23号峰为异绿原酸C、31号峰为木犀草素、35号峰为芹菜素、38号峰为猫眼草黄素、39号峰为青蒿素、40号峰为艾黄素、48号峰为青蒿酸(图3)。图谱显示,异绿原酸A峰面积大且稳定,分离度良好,故对比其保留时间(峰面积),其他峰的相对保留时间(峰面积)=其他峰的保留时间(峰面积)/异绿原酸A的保留时间(峰面积),见表2表3

      编号 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 RSD(%)
      峰1 0.071 0.071 0.071 0.071 0.070 0.070 0.070 0.071 0.070 0.072 0.072 1.060
      峰2 0.075 0.075 0.075 0.075 0.075 0.074 0.075 0.075 0.074 0.075 0.075 0.540
      峰3 0.303 0.308 0.308 0.309 0.309 0.309 0.310 0.311 0.310 0.310 0.309 0.680
      峰4 0.396 0.401 0.401 0.401 0.400 0.401 0.401 0.402 0.402 0.403 0.402 0.450
      峰5 0.418 0.423 0.423 0.424 0.424 0.423 0.424 0.425 0.424 0.425 0.425 0.460
      峰6 0.433 0.437 0.437 0.437 0.436 0.436 0.436 0.437 0.437 0.439 0.438 0.340
      峰7 0.468 0.472 0.472 0.472 0.472 0.472 0.472 0.473 0.473 0.473 0.473 0.300
      峰8 0.662 0.667 0.667 0.667 0.667 0.666 0.667 0.668 0.667 0.668 0.668 0.250
      峰9 0.717 0.723 0.723 0.723 0.723 0.722 0.724 0.724 0.724 0.724 0.724 0.280
      峰10 0.731 0.739 0.738 0.738 0.741 0.737 0.738 0.738 0.738 0.738 0.740 0.340
      峰11 0.794 0.796 0.796 0.796 0.796 0.795 0.797 0.797 0.796 0.797 0.797 0.120
      峰12 0.810 0.815 0.815 0.815 0.809 0.814 0.815 0.815 0.814 0.817 0.817 0.310
      峰13 0.823 0.825 0.825 0.825 0.825 0.824 0.826 0.827 0.825 0.826 0.826 0.130
      峰14 0.912 0.912 0.912 0.913 0.911 0.912 0.914 0.914 0.913 0.913 0.914 0.110
      峰15 0.983 0.982 0.983 0.983 0.982 0.982 0.983 0.985 0.982 0.984 0.983 0.100
      峰16 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0
      峰17 1.038 1.037 1.037 1.037 1.037 1.037 1.037 1.038 1.037 1.038 1.038 0.050
      峰18 1.058 1.056 1.058 1.058 1.060 1.060 1.061 1.062 1.060 1.059 1.059 0.160
      峰19 1.078 1.079 1.078 1.078 1.075 1.074 1.078 1.079 1.075 1.078 1.079 0.170
      峰20 1.133 1.131 1.131 1.131 1.131 1.129 1.130 1.131 1.129 1.132 1.132 0.110
      峰21 1.157 1.154 1.157 1.155 1.155 1.156 1.156 1.157 1.154 1.158 1.156 0.110
      峰22 1.190 1.190 1.189 1.189 1.190 1.192 1.188 1.189 1.187 1.189 1.192 0.130
      峰23 1.199 1.198 1.198 1.198 1.198 1.198 1.199 1.199 1.197 1.198 1.198 0.050
      峰24 1.215 1.214 1.214 1.214 1.213 1.213 1.213 1.215 1.212 1.214 1.214 0.070
      峰25 1.246 1.244 1.244 1.244 1.243 1.242 1.243 1.244 1.242 1.244 1.245 0.100
      峰26 1.307 1.306 1.306 1.307 1.306 1.305 1.306 1.308 1.306 1.306 1.308 0.070
      峰27 1.452 1.453 1.450 1.450 1.450 1.450 1.450 1.453 1.449 1.448 1.449 0.110
      峰28 1.464 1.462 1.463 1.463 1.464 1.463 1.464 1.465 1.461 1.461 1.460 0.110
      峰29 1.497 1.493 1.495 1.495 1.492 1.492 1.495 1.500 1.492 1.494 1.494 0.160
      峰30 1.656 1.655 1.656 1.653 1.648 1.645 1.648 1.649 1.647 1.658 1.659 0.300
      峰31 1.665 1.665 1.672 1.663 1.659 1.657 1.657 1.658 1.656 1.667 1.667 0.320
      峰32 1.718 1.718 1.717 1.716 1.711 1.709 1.710 1.711 1.708 1.719 1.721 0.270
      峰33 1.752 1.752 1.751 1.748 1.743 1.738 1.738 1.740 1.739 1.754 1.756 0.400
      峰34 1.770 1.770 1.769 1.767 1.762 1.758 1.758 1.759 1.758 1.772 1.775 0.360
      峰35 1.833 1.834 1.833 1.830 1.825 1.821 1.821 1.822 1.820 1.835 1.838 0.360
      峰36 1.893 1.894 1.893 1.890 1.884 1.879 1.879 1.880 1.879 1.896 1.900 0.420
      峰37 1.942 1.943 1.942 1.939 1.932 1.927 1.927 1.928 1.928 1.945 1.949 0.430
      峰38 1.950 1.950 1.949 1.946 1.939 1.934 1.934 1.935 1.935 1.953 1.956 0.430
      峰39 2.027 2.027 2.026 2.023 2.015 2.010 2.009 2.011 2.011 2.030 2.034 0.460
      峰40 2.036 2.036 2.032 2.031 2.024 2.019 2.018 2.016 2.020 2.039 2.043 0.470
      峰41 2.102 2.102 2.101 2.097 2.090 2.084 2.084 2.085 2.085 2.105 2.109 0.450
      峰42 2.117 2.116 2.114 2.111 2.103 2.097 2.097 2.097 2.098 2.119 2.122 0.470
      峰43 2.156 2.155 2.153 2.149 2.141 2.135 2.134 2.135 2.136 2.157 2.161 0.490
      峰44 2.226 2.224 2.223 2.219 2.211 2.204 2.204 2.205 2.205 2.226 2.230 0.470
      峰45 2.233 2.232 2.230 2.226 2.218 2.212 2.211 2.213 2.212 2.234 2.238 0.470
      峰46 2.267 2.265 2.263 2.259 2.250 2.243 2.243 2.244 2.244 2.265 2.270 0.490
      峰47 2.329 2.327 2.325 2.321 2.312 2.306 2.304 2.306 2.306 2.328 2.332 0.480
      峰48 2.363 2.362 2.36 2.355 2.346 2.339 2.338 2.340 2.340 2.363 2.367 0.490
      共有峰/总峰 0.837 0.815 0.778 0.821 0.811 0.834 0.864 0.776 0.823 0.806 0.838
      编号 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 RSD(%)
      峰1 0.221 0.718 0.167 0.215 6.001 18.615 0.134 0.037 0.545 0.046 0.033 232.240
      峰2 0.539 2.042 0.539 0.505 11.735 36.124 0.337 0.055 1.571 0.192 0.041 223.380
      峰3 0.065 0.102 0.060 0.131 0.300 0.356 0.050 0.035 0.070 0.032 0.028 100.170
      峰4 0.066 0.061 0.059 0.088 0.121 0.476 0.033 0.015 0.064 0.021 0.026 139.490
      峰5 0.134 0.056 0.104 0.130 0.183 0.339 0.145 0.084 0.080 0.054 0.073 64.820
      峰6 0.391 0.338 0.288 0.344 0.243 0.408 0.321 0.169 0.271 0.131 0.077 39.430
      峰7 0.987 0.201 0.214 0.514 0.385 0.315 0.850 0.405 0.308 0.251 0.482 57.330
      峰8 0.375 0.295 0.217 0.584 0.686 0.903 0.376 0.150 0.338 0.143 0.305 59.310
      峰9 0.533 0.646 0.316 0.766 2.647 4.610 0.201 0.130 0.674 0.112 0.332 139.510
      峰10 0.223 0.058 0.024 0.137 0.259 0.464 0.189 0.008 0.051 0.039 0.023 104.800
      峰11 0.225 0.203 0.202 0.236 0.365 0.952 0.079 0.120 0.145 0.112 0.069 101.160
      峰12 0.119 0.213 0.234 0.245 0.516 0.872 0.116 0.072 0.117 0.055 0.049 105.050
      峰13 0.197 0.181 0.207 0.214 0.376 1.106 0.085 0.107 0.143 0.111 0.061 116.600
      峰14 0.055 0.151 0.081 0.096 0.132 0.242 0.064 0.129 0.067 0.047 0.058 57.120
      峰15 0.125 0.315 0.186 0.226 0.295 0.477 0.109 0.138 0.179 0.062 0.126 58.660
      峰16 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0
      峰17 1.130 0.724 0.667 1.118 0.879 0.891 1.175 0.356 0.943 0.466 0.849 31.770
      峰18 0.104 0.101 0.185 0.115 0.241 0.060 0.089 0.224 0.053 0.108 0.087 51.150
      峰19 0.551 0.465 0.406 0.497 1.441 1.610 0.265 0.566 0.291 0.253 0.229 79.600
      峰20 0.050 0.093 0.222 0.077 0.080 0.171 0.041 0.214 0.047 0.079 0.029 69.220
      峰21 0.086 0.169 0.206 0.138 0.095 0.248 0.085 0.079 0.120 0.066 0.058 50.140
      峰22 0.096 0.098 0.081 0.120 0.130 0.177 0.105 0.050 0.112 0.069 0.078 33.720
      峰23 0.251 0.842 0.321 0.530 0.626 0.594 0.206 0.256 0.418 0.121 0.353 52.610
      峰24 0.237 0.280 0.147 0.286 0.324 0.264 0.244 0.138 0.289 0.122 0.176 30.790
      峰25 0.445 0.565 0.441 0.730 0.725 0.619 0.486 0.121 0.558 0.243 0.306 40.560
      峰26 0.175 0.211 0.243 0.279 0.427 0.832 0.121 0.107 0.199 0.093 0.120 83.960
      峰27 0.050 0.045 0.030 0.090 0.161 0.245 0.056 0.006 0.071 0.028 0.027 95.860
      峰28 0.063 0.243 0.054 0.177 0.170 0.123 0.049 0.036 0.127 0.018 0.080 68.020
      峰29 0.107 0.125 0.092 0.177 0.361 0.428 0.091 0.015 0.109 0.053 0.041 90.430
      峰30 0.080 0.390 0.063 0.298 1.373 1.312 0.065 0.019 0.113 0.026 0.030 148.400
      峰31 0.071 0.216 0.066 0.163 0.244 0.216 0.065 0.023 0.100 0.030 0.158 64.710
      峰32 0.027 0.071 0.019 0.054 0.303 0.210 0.030 0.014 0.032 0.017 0.018 131.430
      峰33 0.160 0.445 0.234 0.380 1.852 2.801 0.146 0.036 0.261 0.087 0.075 151.790
      峰34 0.114 0.280 0.166 0.312 0.777 1.295 0.117 0.019 0.159 0.054 0.055 128.230
      峰35 0.336 1.630 0.399 1.267 6.102 5.469 0.423 0.117 0.603 0.123 0.317 141.670
      峰36 0.070 0.330 0.042 0.260 1.421 1.875 0.063 0.004 0.116 0.013 0.021 167.530
      峰37 0.153 0.999 0.269 0.774 3.799 5.304 0.195 0.088 0.399 0.071 0.106 159.420
      峰38 0.304 0.702 0.221 0.534 3.139 3.099 0.303 0.068 0.400 0.101 0.143 140.610
      峰39 0.090 0.447 0.148 0.331 1.801 2.513 0.123 0.022 0.194 0.042 0.033 159.710
      峰40 0.084 0.285 0.091 0.199 1.102 1.365 0.043 0.076 0.122 0.021 0.016 151.000
      峰41 0.190 0.366 0.232 0.172 1.131 2.101 0.068 0.033 0.226 0.055 0.039 152.190
      峰42 0.157 0.322 0.185 0.229 1.672 2.273 0.121 0.028 0.231 0.047 0.052 155.930
      峰43 0.065 0.137 0.091 0.109 0.535 1.037 0.049 0.017 0.093 0.027 0.027 157.670
      峰44 0.059 0.108 0.068 0.049 0.303 0.650 0.019 0.012 0.062 0.017 0.014 156.080
      峰45 0.110 0.234 0.138 0.113 0.747 1.344 0.045 0.020 0.135 0.033 0.025 153.940
      峰46 0.083 0.114 0.089 0.161 0.705 1.559 0.064 0.024 0.127 0.032 0.049 170.940
      峰47 0.108 0.285 0.176 0.201 1.054 2.412 0.039 0.039 0.161 0.037 0.038 175.050
      峰48 0.389 0.878 0.171 0.854 4.399 5.353 0.419 0.045 0.356 0.065 0.162 155.940
    • 将试剂盒中Trolox溶液按说明书稀释成梯度浓度,加入反应液、不同浓度的Trolox溶液,并设置空白对照。反应结束后,在相应波长下测出测定孔吸光度Ac、空白孔吸光度Ak,自由基清除率(%)=(1−(AcAk)÷Ak)×100%,以浓度(Y)对DPPH自由基清除率(X)进行线性回归,获得标准回归曲线方程,见表4,以标准回归曲线方程计算得出自由基清除能力。

      自由基 标准回归曲线方程 r
      DPPH Y=0.021 8X+0.005 7 0.999 9
      ABTS Y=0.447 6X+0.012 4 0.997 3
    • 取11批供试品溶液(编号:S1~11,按“2.2.2”项下方法制备)各1 ml,加无水甲醇9 ml稀释10倍;对照孔加入供试品100 μl、无水甲醇150 μl,测定孔加入供试品100 μl、DPPH工作液150 μl,空白孔加入供试品100 μl、DPPH工作液150 μl后混匀,避光室温下静置0.5 h,于540 nm波长处测得对照孔吸光度Ad,测定孔吸光度Ac,空白孔吸光度Ak,DPPH自由基清除率(%)=(1−(Ac-Ad)÷Ak)×100%,供试品DPPH自由基清除能力(mmol/L Trolox)=带入标准回归曲线方程所得Trolox的浓度×10,结果见表5。11批青蒿样品中,样品8(编号:S8)清除DPPH自由基的能力最强。

      编号DPPH清除能力ABTS清除能力
      S12.7256.243
      S21.3362.025
      S32.7654.521
      S43.1107.190
      S51.6172.848
      S60.9700.853
      S75.0578.641
      S86.47710.453
      S92.0252.575
      S104.7318.313
      S115.3219.848
    • 取11批供试品溶液(编号:S1~11,按“2.2.2”项下方法制备)各1 ml,加无水甲醇9 ml稀释10倍;对照孔加入供试品100 μl、无水甲醇150 μl,测定孔加入供试品100 μl、ABTS工作液150 μl,空白孔加入无水乙醇100 μl、ABTS工作液150 μl,混匀置于室温反应6 min,于405 nm波长处测得对照孔吸光度Ad,测定孔吸光度Ac,空白孔吸光度Ak,DPPH自由基清除率(%)=(1−(AcAd)÷Ak)×100%,供试品ABTS自由基清除能力(mmol/L Trolox)=代入标准回归曲线方程所得Trolox的浓度×10,结果见表5。11批青蒿样品中,样品8(编号:S8)清除ABTS自由基的能力最强。

    • 表5青蒿抗氧化活性结果和表3的相对峰面积数据导入SIMCA 14.1软件构建PLSR模型。结果显示,峰3~峰8、峰10~峰27、峰29、峰32~峰34、峰38、峰40、峰42~峰43、峰46与DPPH自由基清除率呈正相关;峰3~峰29、峰31~峰35、峰38、峰40、峰42~峰44、峰46、峰48与ABTS自由基清除率呈正相关,结果见图4

      变量投影(VIP)用于评估每个自变量对因变量的重要性,VIP>1表明自变量对因变量的贡献有显著性。筛选出对自由基清除能力呈正相关,且VIP>1的峰。结果显示,青蒿药材样品已明确成分中对DPPH自由基清除能力的贡献大小依次为异绿原酸A(16号峰)>异绿原酸B(15号峰)>滨蒿内酯(14号峰)>异绿原酸C(23号峰);青蒿药材样品已明确成分中对ABTS自由基清除能力的贡献大小依次为异绿原酸A(16号峰)>异绿原酸B(15号峰)>异绿原酸C(23号峰)>滨蒿内酯(14号峰),提示相应成分与青蒿抗氧化能力呈正相关,结果见图5

    • 利用HPLC法测定青蒿药材样品的图谱,能准确、稳定地检测青蒿中的多种有效成分。实验前期研究发现,在乙腈-0.1%磷酸水、甲醇-0.1%磷酸水等流动相中,甲醇-0.1%磷酸水为流动相的分离效果最优,但青蒿素和艾黄素的分离度不佳;随后将水相改为0.2%磷酸水后,分离度得到了提高。研究前期考察了多个波长下各成分的显峰情况,发现在高波长下青蒿素不显峰,故最后选择220 nm为指纹图谱波长,但低波长时显峰较多,在一定程度上影响了几种已知成分的VIP值。

        本研究以11批不同地区青蒿为研究对象,采用HPLC法研究其各共有峰与体外抗氧化能力之间的关系,探究青蒿体外抗氧化作用的药效物质基础。结果显示,异绿原酸A、异绿原酸B、异绿原酸C和滨蒿内酯与自由基清除率呈正相关,提示这些成分是青蒿体外抗氧化的药效物质基础。青蒿素是青蒿中的重要成分,有文献研究发现其具有抗氧化的作用[12],也有文献研究发现其可以激活氧化应激[13],而本研究发现青蒿素的抗氧化作用并不明显。推测原因可能在于本研究所用的是基于清除自由基的抗氧化测试方法,青蒿素可能是通过清除自由基之外的途径起到抗氧化作用[14],仍有待今后深入研究。

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