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中国精品科技期刊2020

夏枯草中16种酚类化合物含量的测定及其与抗氧化活性的相关性分析

罗敏, 何婷, 龚磊, 陈荣祥

罗敏,何婷,龚磊,等. 夏枯草中16种酚类化合物含量的测定及其与抗氧化活性的相关性分析[J]. 食品工业科技,2023,44(1):299−306. doi: 10.13386/j.issn1002-0306.2022030172.
引用本文: 罗敏,何婷,龚磊,等. 夏枯草中16种酚类化合物含量的测定及其与抗氧化活性的相关性分析[J]. 食品工业科技,2023,44(1):299−306. doi: 10.13386/j.issn1002-0306.2022030172.
LUO Min, HE Ting, GONG Lei, et al. Determination of 16 Phenolic Compounds in Prunella vulgaris and Analysis Their Correlation with Antioxidant Activity[J]. Science and Technology of Food Industry, 2023, 44(1): 299−306. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022030172.
Citation: LUO Min, HE Ting, GONG Lei, et al. Determination of 16 Phenolic Compounds in Prunella vulgaris and Analysis Their Correlation with Antioxidant Activity[J]. Science and Technology of Food Industry, 2023, 44(1): 299−306. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022030172.

夏枯草中16种酚类化合物含量的测定及其与抗氧化活性的相关性分析

基金项目: 国家自然科学基金(81760652)。
详细信息
    作者简介:

    罗敏(1997−),女,硕士研究生,研究方向:药用植物质量评价,E-mail:1356080583@qq.com

    通讯作者:

    陈荣祥(1984−),男,博士,副教授,研究方向:药用植物开发与利用,E-mail:chenrongxiang2014@163.com

  • 中图分类号: TS201.2

Determination of 16 Phenolic Compounds in Prunella vulgaris and Analysis Their Correlation with Antioxidant Activity

  • 摘要: 目的:采用液相色谱-串联质谱法(LC-MS/MS)测定24批夏枯草中16种酚类化合物的含量,并采用灰色关联分析和偏最小二乘回归分析方法探讨酚类化合物与抗氧化能力的相关性。方法:夏枯草样品用80%甲醇超声提取,并基于LC-MS/MS建立夏枯草中16种酚类化合物的含量测定方法,以Waters ACQUITY UPLC BEH C18(2.1 mm×100 mm,1.7 μm)色谱柱为固定相,以0.1%甲酸-乙腈为流动相在体积流量为0.4 mL/min条件下进行梯度洗脱,负离子多反应监测模式检测。以DPPH自由基清除能力、ABTS自由基清除能力和铁离子还原能力评价夏枯草的抗氧化活性。结果:16种酚类化合物在各自浓度范围内线性关系良好,相关系数r均大于0.99,精密度、重复性、稳定性和回收率良好。体外抗氧化实验证明夏枯草具有较强的清除ABTS、DPPH自由基和铁离子还原能力,灰色关联分析和偏最小二乘回归分析结果表明迷迭香酸、咖啡酸、芦丁、金丝桃苷、异槲皮苷对于抗氧化的贡献较大。结论:本研究建立了同时测定夏枯草中16种酚类化合物的LC-MS/MS方法,进一步通过相关性分析确定了夏枯草抗氧化的主要活性成分,从而为夏枯草产品的质量控制与开发利用提供科学依据。
    Abstract: Objective: To determine the content of 16 phenolic compounds in 24 batches of Prunella vulgaris by liquid chromatography- tandem mass spectrometry (LC-MS/MS) and to investigate the correlation between phenolic compounds and antioxidant activity by grey relational analysis (GRA) and partial least squares regression (PLSR) analysis. Methods: The sample was extracted with 80% methanol by the ultrasonic extraction. A method for the determination of 16 phenolic compounds in Prunella vulgaris was established by LC-MS/MS. The Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 μm) was selected as the stationary phase. The gradient elution was performed with the mobile phase of 0.1% formic acid-acetonitrile at the flow rate of 0.4 mL/min. The eluted compounds were detected in the negative ion mode with multiple reaction monitoring technology. The antioxidant activities were evaluated by DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and ferric reducing antioxidant power. Results: There were significant linear relationship of 16 phenolic compounds in the range of their concentration with the correlation coefficient (r) greater than 0.99. The precision, repeatability, stability and recovery were good. In vitro antioxidant experiments showed that Prunella vulgaris had strong ability to scavenge ABTS, DPPH free radicals and ferric reduction. The results of GRA and PLSR indicated that rosmarinic acid, caffeic acid, rutin, hyperoside and isoquercitrin were the primary compounds contributing to the antioxidant capacity. Conclusion: A LC-MS/MS method for simultaneous determination of 16 phenolic compounds in Prunella vulgaris was established and the primary antioxidant active components were further investigated by correlation analysis, so as to provide scientific basis for quality control, development and utilization of Prunella vulgaris.
  • 夏枯草(Prunella vulgaris L.)是唇形科夏枯草属多年生草本植物,广泛分布于东亚和欧洲,在我国有悠久的食用和药用历史[1-2]。夏枯草的主要药用和食用部位为其干燥果穗,具有抗氧化、增强免疫、抗病毒、抗炎、降血压和抗肿瘤等作用[3-4],可作为糖尿病的保健食品和药品[5],也可用作汤料和凉茶[6],其花蜜经充分酿造后具有很好的抗结肠炎和调节肠道菌群的作用[7]。夏枯草含有丰富的酚酸、黄酮、三萜酸等化学成分[2, 8],其中酚类化合物是夏枯草重要的活性成分[9]。酚类化合物可抵御紫外线或病原菌的侵袭,具有很好的抗氧化活性,可清除自由基对人体生命造成的损伤[10],目前已经作为天然的抗氧化剂被广泛应用于食品方面[11]。夏枯草中的酚类化合物主要有迷迭香酸、丹参素、咖啡酸等,在《中国药典》2020年版中,迷迭香酸就被作为夏枯草含量测定的质量评价指标[12]。近年的研究也多采用高效液相色谱法测定酚类物质的含量,但是所能测定的指标多集中于迷迭香酸、丹参素、咖啡酸等少数几种化合物[13-14]

    相对传统的液相色谱-紫外检测法,液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS)具有分析速度快、分离效能高、灵敏度高等优点,现被广泛应用于各种复杂化合物的分析,且测定低限可达纳克级[15-17]。如Ozdal等[18]采用LC-MS/MS法研究了土耳其蜂胶提取物中的多酚组成,并鉴定出32种酚类化合物;Schulz等[19]采用LC-MS/MS法定量了黄色番石榴果实酸水解后的23种酚类物质,不仅具有极高的灵敏度,而且还可提供常规方法难以获取的结构信息;罗弘杉等[20]采用液相色谱-高分辨质谱鉴定夏枯草中的酚类化合物,具有较高的分辨率,但未对多组分进行定量分析。

    在色谱分离的基础上,将化学成分和生物活性成分相结合进行相关性分析,可明确不同成分对于生物活性的贡献。灰色关联分析(grey correlation analysis, GRA)和偏最小二乘回归(partial least squares regression, PLSR)分析是较常用的多因素统计方法,GRA将因素间发展的相似性程度作为衡量系统中各因素间的关联程度,即灰色关联度(gray relational degree, GRD),现已被广泛用于解决多因素、多变量之间复杂相互关系的问题;PLSR是一种对主成分分析和多元回归的特征进行泛化和组合的方法,能有效地解决变量间的多重共线性问题[17, 21]。目前对于各种食品的品质调控多采用PLSR或GRA进行评价,并以此计算食品品质的影响程度,进而分析影响因素与综合评分的相关性[22-23]

    因此,本研究采用LC-MS/MS对夏枯草中16种酚类化合物进行定量分析,采用DPPH、ABTS自由基清除能力和铁离子还原能力(ferric reducing antioxidant power, FRAP)对夏枯草的抗氧化活性进行评价,并进一步采用GRA、PLSR法对24批夏枯草中16种酚类化合物的相关性进行分析,以此探讨夏枯草抗氧化的物质基础,为全面提高夏枯草的质量控制水平及其产品开发和应用提供参考。

    夏枯草样本分别采自广东、河南等9个省份,其详细信息见表1。样本经遵义医科大学顾丁副教授鉴定为唇形科植物夏枯草(Prunella vulgaris L)的干燥果穗。

    表  1  24批夏枯草样品信息
    Table  1.  Information of 24 batches of Prunella vulgaris
    样品编号产地 样品编号产地 样品编号产地
    S1广东深圳 S9江苏常州 S17湖北襄阳
    S2安徽亳州S10河南周口S18浙江嘉兴
    S3河南郑州S11安徽安庆S19湖北咸宁
    S4湖北荆州S12河南漯河S20河北沧州
    S5安徽合肥S13安徽黄山S21安徽宣城
    S6内蒙包头S14广东湛江S22河北承德
    S7河南新乡S15广东佛山S23广东广州
    S8河北石家庄S16河北保定S24安徽池州
    下载: 导出CSV 
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    对羟基苯甲酸、异槲皮苷、七叶内酯 Ark Pharm公司;对羟基肉桂酸、2,5-二羟基苯甲酸 Adamas-beta公司;迷迭香酸、绿原酸、咖啡酸、3,4-二羟基苯甲酸、金丝桃苷、阿魏酸、水杨酸、芦丁 上海阿拉丁生化科技股份有限公司;异迷迭香酸苷 成都麦德生科技有限公司;丹参素 辰光生物有限公司;3,4-二羟基苯甲醛 北京伊诺凯科技有限公司;所有对照品的纯度≥97%;乙腈 质谱纯,霍尼韦尔贸易有限公司;室验用水 屈臣氏;其余试剂为分析纯。

    I-class+XevoTQ-S超高效液相色谱-三重四极杆质谱仪(配MassLynx V.4.1软件) 美国Waters;SB-5200DT型超声波清洗机 宁波新芝生物科技;ME104E电子天平 美国梅特勒-托利多。

    将夏枯草粉碎,过60目筛。准确称取1 g样品粉末,加40 mL 80%的甲醇在室温条件下超声提取30 min,提取完成后将提取液置于−20 ℃条件下静置1 h,吸取上清液,用0.22 μm微孔滤膜过滤后进行色谱分析和抗氧化能力测定。

    精密称取16种夏枯草对照品,分别加入甲醇溶解定容至10 mL,配制成浓度为1 mg/mL的标准品溶液,−20 ℃保存备用。使用时用80%甲醇将其稀释成所需的混合对照品工作溶液。

    采用Waters ACQUITY UPLC BEH C18(2.1 mm×100 mm,1.7 μm)色谱柱,采用0.1%甲酸(A)-乙腈(B)作为流动相在体积流量为0.4 mL/min下进行梯度洗脱,洗脱程序为0~15 min,95%~70% A;15~19.5 min,70%~50% A;19.5~20.5 min,50%~5% A;20.5~21.5 min,5% A;21.5~22 min,5%~95% A;22~25 min,95% A。柱温为45 ℃;进样器温度为12 ℃;进样量为1 μL。

    采用电喷雾离子源在负离子及多反应监测(multiple reaction monitoring, MRM)模式下检测16种酚类化合物,并采用两组离子对分别对16种酚类化合物进行定量和定性分析,采用外标法进行定量。毛细管电压为3 kV,蒸发温度为500 ℃、气流量为750 L/Hr。

    取醋酸盐缓冲溶液(pH3.6)、10 mmol/L TPTZ溶液、20 mmol/L FeCl3溶液加入到600 μL稀释后的样品溶液中混匀,暗反应30 min后于593 nm波长处测定样品吸光度。空白组中样品溶液的吸光度测定采用80%的甲醇代替。以Trolox为标准品绘制标准曲线,线性回归方程和相关系数分别为Y=0.0258X+0.0645和r=0.9997,其中,Y为吸光度(Au),X为Trolox浓度(mg/L)。所有样品测量结果均以Trolox当量表示(mg TE/g DW)。

    ABTS工作液的配制:准确称取ABTS试剂0.0282 g,用蒸馏水溶解定容至7.5 mL,再加入7.5 mL 2.45 mmol/L过硫酸钾水溶液,在室温下避光放置8~12 h后用纯水稀释20倍后备用。取200 μL稀释100倍后的样品溶液加入到0.4 mL ABTS工作液中,暗反应30 min后于734 nm波长处测定样品吸光度。空白组中样品溶液的吸光度测定以80%甲醇代替,以Trolox为标准品绘制标准曲线,线性回归方程和相关系数分别为Y=−0.00901X+0.5311和r=0.9998,其中Y为吸光度(Au),X为Trolox浓度(mg/L)。所有样品测量结果的表示同“1.2.4.1”项。

    取200 μL稀释30倍后的样品溶液加入到400 μL 0.14 mol/L的DPPH溶液中,混匀,暗反应30 min后于517 nm波长处测定样品吸光度。空白组中样品溶液的吸光度测定以及所有样品测量结果的表示同“1.2.4.1”项。以Trolox为标准品绘制标准曲线,线性回归方程和相关系数分别为Y=−0.0155X+0.8361和r=0.9993。其中Y为吸光度(Au),X为Trolox浓度(mg/L)。

    以16种酚类化合物含量和DPPH、ABTS自由基清除能力和FRAP效应值分别作为自变量和因变量。在判别系数设为0.5的情况下,计算GRD值,GRD越高,相应的酚类化合物抗氧化活性越大。

    采用16种酚类化合物的含量作为自变量,DPPH、ABTS和FRAP值作为因变量。将数据导入软件SIMCA 14.1中进行PLSR分析,得出各自的CoeffCS值和VIP值,以分析自变量与因变量的关系。

    采用Waters公司的MassLynx V4.1软件进行数据采集;采用Excel进行数据分析;采用Origin 2018版以及SIMCA 14.1版软件进行绘图。

    对16种化合物的标准品进行锥孔电压、碰撞能量等条件的优化,优化结果见表2。标准品图见图1A,除丹参素、迷迭香酸和异迷迭香酸苷浓度为5 mg/L外,其余化合物浓度均为0.5 mg/L。夏枯草样品图见图1B

    表  2  16种主要成分的保留时间和质谱参数
    Table  2.  Retention time and mass parameters of the 16 components
    序号化合物保留时间
    (min)
    母离子
    (m/z)
    子离子
    (m/z)
    锥孔电压
    (V)
    碰撞能量
    (eV)
    1丹参素1.61197.2135.1*/179.12020
    23,4-二羟基苯甲酸1.83153.1109.1*/—3012
    33,4-二羟基苯甲醛2.67137.1108.1*/90.01520
    4对羟基苯甲酸2.68137.193.1*/ —4512
    52,5-二羟基苯甲酸2.78153.1109.1*/ —3012
    6绿原酸3.19353.1191.1*/179.11215
    7七叶内酯3.47177.1133.1*/149.13518
    8咖啡酸3.62178.1135.1*/117.13816
    9对羟基肉桂酸5.2163.1119.1*/115.11215
    10阿魏酸6.26193.1134.1*/178.11515
    11芦丁7.04609.6301.3*/271.23035
    12金丝桃苷7.05463.4301.3*/300.34020
    13异槲皮苷7.29463.4301.3*/271.24020
    14水杨酸7.64137.193.1*/ —2015
    15异迷迭香酸苷8.08521.5359.3*/179.12020
    16迷迭香酸9.42359.3197.1*/179.1188
    注:*代表定量离子对;—代表未检测到离子信息。
    下载: 导出CSV 
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    图  1  混合对照品(A)和样品(B)提取离子流色谱图
    注:1:丹参素;2:3,4-二羟基苯甲酸;3:3,4-二羟基苯甲醛;4:对羟基苯甲酸;5:2,5-二羟基苯甲酸;6:绿原酸;7:七叶内酯;8:咖啡酸;9:对羟基肉桂酸;10:阿魏酸;11:芦丁;12:金丝桃苷;13:异槲皮苷;14:水杨酸;15:异迷迭香酸苷;16:迷迭香酸。
    Figure  1.  The extract ion chromatograms of mixed reference (A) and testing sample (B)

    分别精密吸取“1.2.2”项下的对照品溶液配制成不同浓度的混标,按“1.2.3”项下的色谱条件进样分析,记录测定后16种酚类化合物的峰面积。以对照品的浓度(mg/L)为横坐标,各待测成分色谱峰峰面积为纵坐标绘制标准曲线,计算回归方程。结果显示,16种酚类成分在各自的线性范围内,线性关系良好(r>0.99)。检出限(S/N=3)在2.80~9.47 ng/mL之间,其回归方程以及相关系数见表3

    表  3  方法学考察
    Table  3.  Method validation of the proposed method
    序号组分线性范围
    (mg/L)
    线性方程相关系数
    r
    LOD
    (ng/mL)
    平均加标回收率
    (%)
    RSD(%)
    精密度稳定性重复性回收率
    1咖啡酸0.2~10Y=281949.37X+5281.0590.99644.4299.401.031.241.311.56
    2对羟基苯甲酸0.05~2Y=3177.54X+81.2730.99826.3699.650.834.484.405.50
    33,4-二羟基苯甲酸0.05~2Y=125185.86X+3446.9980.99809.1598.500.430.690.761.70
    43,4-二羟基苯甲醛0.05~2Y=40216.21X+448.460.99889.1598.650.500.910.342.76
    52,5-二羟基苯甲酸0.005~0.2Y=73228.37X+140.970.99894.8195.312.245.065.465.13
    6水杨酸0.01~0.5Y=317026.83X+1976.850.99877.7996.981.314.905.364.72
    7芦丁0.2~40Y=20705.10X+1078.380.99669.2699.101.563.763.732.61
    8对羟基肉桂酸0.01~0.5Y=500113.23X+1136.140.99902.8097.811.634.504.735.02
    9七叶内酯0.01~2Y=213825.34X+2134.230.99937.3196.711.462.321.982.64
    10阿魏酸0.01~0.5Y=23305.22X+41.590.99988.8896.761.494.705.716.24
    11丹参素2~50Y=17645.89X+4551.480.99547.43102.340.643.392.613.21
    12金丝桃苷0.01~10Y=38262.69X+1803.650.99937.9198.011.033.743.153.45
    13迷迭香酸5~500Y=4924.67X−5552.720.99899.47103.740.41.050.931.76
    14绿原酸0.02~5Y=170804.82X+1108.710.99213.9898.491.002.341.691.54
    15异槲皮苷0.01~10Y=101301.31X+4147.820.99648.0898.891.013.493.205.02
    16异迷迭香酸苷0.5~50Y=13823.36X+6247.780.99966.8998.921.121.771.482.28
    下载: 导出CSV 
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    称取编号为S14的夏枯草粉末约1 g,按“1.2.1 样品制备”方法平行制备6份供试品溶液,按上述色谱和质谱条件,测定并记录夏枯草中16种化合物的峰面积,计算RSD值,结果显示RSD值≤5.71%。

    精密量取标准品储备溶液并将其配制成0.5 mg/L的工作液(其中,异迷迭香酸苷、丹参素和迷迭香酸浓度为5 mg/L),按上述质谱和色谱条件,连续进样6次,进样量1 μL,测定并记录16种化合物的峰面积,计算RSD值,结果显示RSD值≤2.24%。

    称取编号为S14的夏枯草粉末约1 g,按“1.2.1 样品制备”方法制备供试品溶液,按上述测试条件在24 h(0、4、8 、12、16、24 h)内测定16种化合物的稳定性,记录夏枯草中16种化合物的峰面积变化并计算RSD值。结果显示24 h内RSD值≤5.06%。

    取同一批含量已知的夏枯草(S6)粉末3份,每份约1 g,按照含量1:1准确加入16种化合物,按“1.2.1 样品制备”方法制备供试品溶液,按上述测试条件进行测定,考察回收率,平均加标回收率在95.31%~103.74%之间,其RSD值≤6.24%。方法学考察结果见表3

    分别称取夏枯草样品粉末,按“1.2.1 样品制备”方法制备供试品溶液,进样,按上述测试条件测定夏枯草中16种酚类化合物并记录各种化合物的峰面积,计算其含量,结果见表4

    表  4  24批夏枯草样品中16种酚类化合物含量测定结果(mg/100 g)
    Table  4.  The contents of 16 phenolic compounds in 24 batches of Prunella vulgaris (mg/100 g)
    编号七叶
    内酯
    对羟基苯甲酸3,4-二羟基苯甲醛2,5-二羟基苯甲酸丹参素水杨酸迷迭
    香酸
    咖啡酸阿魏酸异迷迭香酸苷绿原酸3,4-二羟基苯甲酸金丝
    桃苷
    异槲
    皮苷
    芦丁对羟基肉桂酸
    S10.931.871.660.1555.020.14515.6111.090.3565.801.541.236.322.8819.460.20
    S21.161.441.670.1938.790.23769.0515.320.3680.420.692.3821.1012.2931.720.25
    S31.031.541.710.1330.360.21745.2113.180.3284.740.522.3617.2210.4926.480.24
    S40.822.031.770.1259.240.14543.4610.520.2384.161.161.182.651.2414.240.19
    S51.371.991.970.1026.440.28912.2914.500.2948.950.521.7013.609.3535.890.18
    S62.572.542.950.1935.990.301036.7323.560.5082.290.693.4622.5415.7350.480.35
    S70.220.740.670.0318.580.07317.074.670.1593.630.410.700.430.272.760.13
    S81.111.311.710.1530.080.21712.5612.840.3276.120.501.968.716.0125.970.26
    S91.632.102.430.2034.740.27893.3520.240.55115.781.122.6310.036.6238.280.35
    S101.281.311.820.1528.880.23624.5412.620.3082.800.401.966.154.3922.200.26
    S110.932.022.340.1531.130.22683.8814.910.4157.280.642.423.082.0820.540.28
    S120.150.870.600.0317.490.07281.284.210.0778.280.120.600.260.152.570.11
    S131.792.402.650.2337.580.30935.5222.470.64120.671.132.8611.296.9834.270.39
    S140.931.561.610.1224.350.20662.2210.630.2148.820.421.689.776.0822.360.19
    S151.321.741.880.1635.850.22674.6613.980.4492.140.822.202.971.9123.420.31
    S161.062.122.070.1249.250.14477.758.900.3676.781.301.161.950.7612.920.19
    S170.971.451.470.0918.780.14332.787.710.2870.790.331.510.400.286.500.23
    S181.651.982.310.2445.920.28883.2323.840.4292.111.392.699.415.5238.640.36
    S191.482.092.240.1734.010.25683.0615.090.3392.710.892.437.124.2728.250.33
    S200.971.331.470.1116.830.13230.317.450.2444.730.361.420.060.053.820.20
    S211.092.142.510.1434.700.28522.3513.600.5562.550.812.481.780.6010.670.33
    S221.362.692.360.1418.800.21417.9010.180.42101.070.472.500.520.359.300.35
    S231.021.401.530.1224.550.17600.6210.970.3069.280.481.652.571.6818.520.22
    S240.201.220.730.0216.240.05205.973.300.0964.300.130.590.270.172.920.07
    下载: 导出CSV 
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    24批样品中化合物的含量差异很大。其中,迷迭香酸、异迷迭香酸苷含量最高,丹参素次之,其含量分别为205.97~1036.73 mg/100 g、44.73~120.67 mg/100 g和16.24~59.24 mg/100 g。刘月新等[24]的研究表明,夏枯草在不同生长阶段其化学成分含量不同,迷迭香酸和异迷迭香酸苷在枯萎期含量明显增加。刘光敏等[25]的研究指出同一品种夏枯草药材因产地不同其酚类成分含量也有较大差异。因此,样品间的差异不仅与产地有关,而且受采收时间影响。

    DPPH、ABTS自由基清除能力和铁离子还原能力结果见表5。在24批夏枯草样品中,DPPH、ABTS自由基清除能力和铁离子还原能力的范围分别为7.53~43.83 mg TE/g DW、10.75~34.93 mg TE/g DW和8.53~47.76 mg TE/g DW。其中,抗氧化能力最好的夏枯草样品为S6,较差的样品为S7和S24。

    表  5  24批夏枯草抗氧化能力结果
    Table  5.  The antioxidant capacity in 24 batches of Prunella vulgaris
    样品
    编号
    DPPH
    (mg TE/g DW)
    ABTS
    (mg TE/g DW)
    铁离子还原能力
    (mg TE/g DW)
    S128.0624.4924.92
    S233.1831.3134.99
    S332.0128.4833.09
    S414.2724.6525.47
    S536.3831.4039.19
    S643.8334.9347.76
    S712.6310.758.53
    S831.6326.9229.17
    S936.8431.8741.48
    S1027.4622.7624.89
    S1132.6225.5229.56
    S1213.1712.309.13
    S1337.0132.9044.44
    S1434.0430.1435.44
    S1530.3527.4632.73
    S1617.6822.2824.57
    S179.2215.4714.45
    S1835.4232.3945.39
    S1920.8627.1733.53
    S207.8714.9313.81
    S2115.1522.2023.71
    S2212.6716.6916.43
    S2318.0724.2229.03
    S247.5312.309.43
    下载: 导出CSV 
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    利用GRA分析夏枯草中16种酚类化合物含量与抗氧化活性之间的相关性,以评价这些成分的活性贡献。GRD值用来描述成分与抗氧化活性之间的关系,GRD大于0.6说明化合物与抗氧化活性之间具有相关性,GRD大于等于0.8说明相关性较强[26-27]。16种酚类化合物与抗氧化活性相关性的GRD值如表6所示,16种化合物的GRD值均大于0.6,可见这些化合物均和抗氧化活性相关。其中,迷迭香酸与DPPH、ABTS、FRAP的关联度均大于0.8,芦丁和DPPH的关联度大于0.8,咖啡酸、芦丁和FRAP的关联度大于0.8,它们具有相对较高的GRD值可以认为是夏枯草中关键的抗氧化成分。

    表  6  GRA结果
    Table  6.  The results of GRA
    DPPHGRDABTSGRDFRAPGRD
    迷迭香酸0.849迷迭香酸0.833迷迭香酸0.885
    芦丁0.8142,5-二羟基苯甲酸0.797咖啡酸0.836
    水杨酸0.767水杨酸0.792芦丁0.800
    咖啡酸0.764咖啡酸0.783水杨酸0.799
    3,4-二羟基苯甲酸0.7623,4-二羟基苯甲醛0.7442,5-二羟基苯甲酸0.794
    2,5-二羟基苯甲酸0.7363,4-二羟基苯甲酸0.7273,4-二羟基苯甲酸0.772
    3,4-二羟基苯甲醛0.721对羟基肉桂酸0.7263,4-二羟基苯甲醛0.763
    金丝桃苷0.718芦丁0.712七叶内酯0.749
    对羟基肉桂酸0.715对羟基苯甲酸0.712对羟基肉桂酸0.744
    异槲皮苷0.713阿魏酸0.711阿魏酸0.739
    阿魏酸0.711七叶内酯0.690对羟基苯甲酸0.698
    七叶内酯0.706绿原酸0.674绿原酸0.688
    对羟基苯甲酸0.678异迷迭香酸苷0.643异迷迭香酸苷0.675
    丹参素0.673金丝桃苷0.633金丝桃苷0.669
    异迷迭香酸苷0.661异槲皮苷0.619异槲皮苷0.667
    绿原酸0.657丹参素0.614丹参素0.650
    下载: 导出CSV 
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    采用PLSR分析DPPH、ABTS、FRAP法与夏枯草中16种酚类化合物的相关性,以16种酚类化合物含量为X变量,以DPPH、ABTS、FRAP值为Y变量进行相关性分析,结果见图2。CoeffCS系数的正负表示不同化合物同抗氧化活性正相关或负相关,VIP值代表相关性强弱。从结果可以看出,对3种抗氧化活性均呈正相关且VIP值大于1.0的化合物有迷迭香酸、芦丁、金丝桃苷、异槲皮苷、咖啡酸、水杨酸、2,5-二羟基苯甲酸。

    图  2  16种化合物的偏最小二乘回归相关性分析CoeffCS (A)和VIP (B)图
    Figure  2.  Correlation coeffCS (A) and VIP (B) values of the 16 compounds analyzed by PLSR

    大量研究表明,酚类化合物是食用/药用植物抗氧化的主要来源[28-29]。本研究结合GRA与PLSR相关性分析可知,夏枯草中16种酚类化合物均具有抗氧化活性,GRD值均大于0.6,且GRA与PLSR相关性分析均表明迷迭香酸抗氧化能力最好。

    综上所述,本研究成功运用LC-MS/MS法同时测定夏枯草中16种酚类化合物的含量,该方法线性关系良好(r>0.99),灵敏度较高。24批夏枯草中不同酚类化合物含量在0.02~1036.73 mg/100 g之间,其中,迷迭香酸含量最高,为205.97~1036.73 mg/100 g。进一步采用GRA和PLSR分析了化学成分与DPPH、ABTS自由基清除能力和铁离子还原能力的相关性。GRA结果表明16种酚类化合物与抗氧化活性的关联系数均大于0.6,均与抗氧化活性相关。PLSR的结果表明迷迭香酸、芦丁、金丝桃苷、异槲皮苷、咖啡酸等化合物对抗氧化活性的贡献较大。本研究不仅测定了夏枯草中主要化合物的含量,而且明确了主要化学成分对抗氧化的贡献,可为夏枯草质量控制与开发利用提供参考。

  • 图  1   混合对照品(A)和样品(B)提取离子流色谱图

    注:1:丹参素;2:3,4-二羟基苯甲酸;3:3,4-二羟基苯甲醛;4:对羟基苯甲酸;5:2,5-二羟基苯甲酸;6:绿原酸;7:七叶内酯;8:咖啡酸;9:对羟基肉桂酸;10:阿魏酸;11:芦丁;12:金丝桃苷;13:异槲皮苷;14:水杨酸;15:异迷迭香酸苷;16:迷迭香酸。

    Figure  1.   The extract ion chromatograms of mixed reference (A) and testing sample (B)

    图  2   16种化合物的偏最小二乘回归相关性分析CoeffCS (A)和VIP (B)图

    Figure  2.   Correlation coeffCS (A) and VIP (B) values of the 16 compounds analyzed by PLSR

    表  1   24批夏枯草样品信息

    Table  1   Information of 24 batches of Prunella vulgaris

    样品编号产地 样品编号产地 样品编号产地
    S1广东深圳 S9江苏常州 S17湖北襄阳
    S2安徽亳州S10河南周口S18浙江嘉兴
    S3河南郑州S11安徽安庆S19湖北咸宁
    S4湖北荆州S12河南漯河S20河北沧州
    S5安徽合肥S13安徽黄山S21安徽宣城
    S6内蒙包头S14广东湛江S22河北承德
    S7河南新乡S15广东佛山S23广东广州
    S8河北石家庄S16河北保定S24安徽池州
    下载: 导出CSV

    表  2   16种主要成分的保留时间和质谱参数

    Table  2   Retention time and mass parameters of the 16 components

    序号化合物保留时间
    (min)
    母离子
    (m/z)
    子离子
    (m/z)
    锥孔电压
    (V)
    碰撞能量
    (eV)
    1丹参素1.61197.2135.1*/179.12020
    23,4-二羟基苯甲酸1.83153.1109.1*/—3012
    33,4-二羟基苯甲醛2.67137.1108.1*/90.01520
    4对羟基苯甲酸2.68137.193.1*/ —4512
    52,5-二羟基苯甲酸2.78153.1109.1*/ —3012
    6绿原酸3.19353.1191.1*/179.11215
    7七叶内酯3.47177.1133.1*/149.13518
    8咖啡酸3.62178.1135.1*/117.13816
    9对羟基肉桂酸5.2163.1119.1*/115.11215
    10阿魏酸6.26193.1134.1*/178.11515
    11芦丁7.04609.6301.3*/271.23035
    12金丝桃苷7.05463.4301.3*/300.34020
    13异槲皮苷7.29463.4301.3*/271.24020
    14水杨酸7.64137.193.1*/ —2015
    15异迷迭香酸苷8.08521.5359.3*/179.12020
    16迷迭香酸9.42359.3197.1*/179.1188
    注:*代表定量离子对;—代表未检测到离子信息。
    下载: 导出CSV

    表  3   方法学考察

    Table  3   Method validation of the proposed method

    序号组分线性范围
    (mg/L)
    线性方程相关系数
    r
    LOD
    (ng/mL)
    平均加标回收率
    (%)
    RSD(%)
    精密度稳定性重复性回收率
    1咖啡酸0.2~10Y=281949.37X+5281.0590.99644.4299.401.031.241.311.56
    2对羟基苯甲酸0.05~2Y=3177.54X+81.2730.99826.3699.650.834.484.405.50
    33,4-二羟基苯甲酸0.05~2Y=125185.86X+3446.9980.99809.1598.500.430.690.761.70
    43,4-二羟基苯甲醛0.05~2Y=40216.21X+448.460.99889.1598.650.500.910.342.76
    52,5-二羟基苯甲酸0.005~0.2Y=73228.37X+140.970.99894.8195.312.245.065.465.13
    6水杨酸0.01~0.5Y=317026.83X+1976.850.99877.7996.981.314.905.364.72
    7芦丁0.2~40Y=20705.10X+1078.380.99669.2699.101.563.763.732.61
    8对羟基肉桂酸0.01~0.5Y=500113.23X+1136.140.99902.8097.811.634.504.735.02
    9七叶内酯0.01~2Y=213825.34X+2134.230.99937.3196.711.462.321.982.64
    10阿魏酸0.01~0.5Y=23305.22X+41.590.99988.8896.761.494.705.716.24
    11丹参素2~50Y=17645.89X+4551.480.99547.43102.340.643.392.613.21
    12金丝桃苷0.01~10Y=38262.69X+1803.650.99937.9198.011.033.743.153.45
    13迷迭香酸5~500Y=4924.67X−5552.720.99899.47103.740.41.050.931.76
    14绿原酸0.02~5Y=170804.82X+1108.710.99213.9898.491.002.341.691.54
    15异槲皮苷0.01~10Y=101301.31X+4147.820.99648.0898.891.013.493.205.02
    16异迷迭香酸苷0.5~50Y=13823.36X+6247.780.99966.8998.921.121.771.482.28
    下载: 导出CSV

    表  4   24批夏枯草样品中16种酚类化合物含量测定结果(mg/100 g)

    Table  4   The contents of 16 phenolic compounds in 24 batches of Prunella vulgaris (mg/100 g)

    编号七叶
    内酯
    对羟基苯甲酸3,4-二羟基苯甲醛2,5-二羟基苯甲酸丹参素水杨酸迷迭
    香酸
    咖啡酸阿魏酸异迷迭香酸苷绿原酸3,4-二羟基苯甲酸金丝
    桃苷
    异槲
    皮苷
    芦丁对羟基肉桂酸
    S10.931.871.660.1555.020.14515.6111.090.3565.801.541.236.322.8819.460.20
    S21.161.441.670.1938.790.23769.0515.320.3680.420.692.3821.1012.2931.720.25
    S31.031.541.710.1330.360.21745.2113.180.3284.740.522.3617.2210.4926.480.24
    S40.822.031.770.1259.240.14543.4610.520.2384.161.161.182.651.2414.240.19
    S51.371.991.970.1026.440.28912.2914.500.2948.950.521.7013.609.3535.890.18
    S62.572.542.950.1935.990.301036.7323.560.5082.290.693.4622.5415.7350.480.35
    S70.220.740.670.0318.580.07317.074.670.1593.630.410.700.430.272.760.13
    S81.111.311.710.1530.080.21712.5612.840.3276.120.501.968.716.0125.970.26
    S91.632.102.430.2034.740.27893.3520.240.55115.781.122.6310.036.6238.280.35
    S101.281.311.820.1528.880.23624.5412.620.3082.800.401.966.154.3922.200.26
    S110.932.022.340.1531.130.22683.8814.910.4157.280.642.423.082.0820.540.28
    S120.150.870.600.0317.490.07281.284.210.0778.280.120.600.260.152.570.11
    S131.792.402.650.2337.580.30935.5222.470.64120.671.132.8611.296.9834.270.39
    S140.931.561.610.1224.350.20662.2210.630.2148.820.421.689.776.0822.360.19
    S151.321.741.880.1635.850.22674.6613.980.4492.140.822.202.971.9123.420.31
    S161.062.122.070.1249.250.14477.758.900.3676.781.301.161.950.7612.920.19
    S170.971.451.470.0918.780.14332.787.710.2870.790.331.510.400.286.500.23
    S181.651.982.310.2445.920.28883.2323.840.4292.111.392.699.415.5238.640.36
    S191.482.092.240.1734.010.25683.0615.090.3392.710.892.437.124.2728.250.33
    S200.971.331.470.1116.830.13230.317.450.2444.730.361.420.060.053.820.20
    S211.092.142.510.1434.700.28522.3513.600.5562.550.812.481.780.6010.670.33
    S221.362.692.360.1418.800.21417.9010.180.42101.070.472.500.520.359.300.35
    S231.021.401.530.1224.550.17600.6210.970.3069.280.481.652.571.6818.520.22
    S240.201.220.730.0216.240.05205.973.300.0964.300.130.590.270.172.920.07
    下载: 导出CSV

    表  5   24批夏枯草抗氧化能力结果

    Table  5   The antioxidant capacity in 24 batches of Prunella vulgaris

    样品
    编号
    DPPH
    (mg TE/g DW)
    ABTS
    (mg TE/g DW)
    铁离子还原能力
    (mg TE/g DW)
    S128.0624.4924.92
    S233.1831.3134.99
    S332.0128.4833.09
    S414.2724.6525.47
    S536.3831.4039.19
    S643.8334.9347.76
    S712.6310.758.53
    S831.6326.9229.17
    S936.8431.8741.48
    S1027.4622.7624.89
    S1132.6225.5229.56
    S1213.1712.309.13
    S1337.0132.9044.44
    S1434.0430.1435.44
    S1530.3527.4632.73
    S1617.6822.2824.57
    S179.2215.4714.45
    S1835.4232.3945.39
    S1920.8627.1733.53
    S207.8714.9313.81
    S2115.1522.2023.71
    S2212.6716.6916.43
    S2318.0724.2229.03
    S247.5312.309.43
    下载: 导出CSV

    表  6   GRA结果

    Table  6   The results of GRA

    DPPHGRDABTSGRDFRAPGRD
    迷迭香酸0.849迷迭香酸0.833迷迭香酸0.885
    芦丁0.8142,5-二羟基苯甲酸0.797咖啡酸0.836
    水杨酸0.767水杨酸0.792芦丁0.800
    咖啡酸0.764咖啡酸0.783水杨酸0.799
    3,4-二羟基苯甲酸0.7623,4-二羟基苯甲醛0.7442,5-二羟基苯甲酸0.794
    2,5-二羟基苯甲酸0.7363,4-二羟基苯甲酸0.7273,4-二羟基苯甲酸0.772
    3,4-二羟基苯甲醛0.721对羟基肉桂酸0.7263,4-二羟基苯甲醛0.763
    金丝桃苷0.718芦丁0.712七叶内酯0.749
    对羟基肉桂酸0.715对羟基苯甲酸0.712对羟基肉桂酸0.744
    异槲皮苷0.713阿魏酸0.711阿魏酸0.739
    阿魏酸0.711七叶内酯0.690对羟基苯甲酸0.698
    七叶内酯0.706绿原酸0.674绿原酸0.688
    对羟基苯甲酸0.678异迷迭香酸苷0.643异迷迭香酸苷0.675
    丹参素0.673金丝桃苷0.633金丝桃苷0.669
    异迷迭香酸苷0.661异槲皮苷0.619异槲皮苷0.667
    绿原酸0.657丹参素0.614丹参素0.650
    下载: 导出CSV
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  • 收稿日期:  2022-03-14
  • 网络出版日期:  2022-11-02
  • 刊出日期:  2022-12-31

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