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牛蒡叶活性成分快速提取及对木腐菌抑制效果

荣宾宾 徐国祺 王立海

荣宾宾, 徐国祺, 王立海. 牛蒡叶活性成分快速提取及对木腐菌抑制效果[J]. 北京林业大学学报, 2017, 39(5): 109-116. doi: 10.13332/j.1000-1522.20170010
引用本文: 荣宾宾, 徐国祺, 王立海. 牛蒡叶活性成分快速提取及对木腐菌抑制效果[J]. 北京林业大学学报, 2017, 39(5): 109-116. doi: 10.13332/j.1000-1522.20170010
RONG Bin-bin, XU Guo-qi, WANG Li-hai. from Arctium lappa leaves and its anti-fungal properties of wood rot fungi[J]. Journal of Beijing Forestry University, 2017, 39(5): 109-116. doi: 10.13332/j.1000-1522.20170010
Citation: RONG Bin-bin, XU Guo-qi, WANG Li-hai. from Arctium lappa leaves and its anti-fungal properties of wood rot fungi[J]. Journal of Beijing Forestry University, 2017, 39(5): 109-116. doi: 10.13332/j.1000-1522.20170010

牛蒡叶活性成分快速提取及对木腐菌抑制效果

doi: 10.13332/j.1000-1522.20170010
基金项目: 

国家自然科学基金项目 31500470

高等学校博士学科点专项科研基金项目 20130062110001

详细信息
    作者简介:

    荣宾宾。主要研究方向:木材防腐。Email: 1570096355@qq.com   地址: 150040  黑龙江省哈尔滨市香坊区和兴路26号东北林业大学工程技术学院

    责任作者:

    王立海,教授,博士生导师。主要研究方向:森工产品开发与检测。Email: lihaiwang@yahoo.com   地址:同上

  • 中图分类号: Q946;R284.2

from Arctium lappa leaves and its anti-fungal properties of wood rot fungi

  • 摘要: 研究了牛蒡叶中抑制木腐菌活性成分的快速提取工艺及其提取物对木材腐朽菌的抑制性能。在以彩绒革盖菌为提取物抑菌效果指示菌的提取预实验以及以提取率为指标的单因素实验基础上,采用响应曲面法对牛蒡叶中活性成分进行优化提取,确定最优提取方式和对木材腐朽菌(彩绒革盖菌和密粘褶菌)的抑制能力,并通过Accela U-HPLC高速液相色谱系统分离和鉴定优化条件下提取物中的活性成分。结果表明:在预实验中,酸性水提取物的抑菌效果要优于其他方式;经过响应曲面四因素三水平优化后,在液料比30 mL/g、提取温度52 ℃、提取时间77 min和溶剂pH值2.0条件下,理论提取率达到45.81%,经验证此条件下的平均提取率为44.86%,与模型理论值差异为-2.08%;此条件下的提取物,比预实验中采用酸性水方式获得的提取物抑制白腐菌的效果提高了16.47%;在此条件下获取的提取物,经U-HPLC-MS分离并鉴定出12种具有抑菌活性的成分,分别为有机酸类(奎宁酸、原儿茶酸、异绿原酸A、异绿原酸B、异绿原酸C、二聚绿原酸、绿原酸、4, 5-二咖啡酰奎尼酸)、黄酮类(牛蒡酮-a)、苷类(芦丁、5′-Propanediolmatairesinoside)、萜类(β-桉叶醇)。经验证,响应曲面优化后得到的提取物对彩绒革盖菌和密粘褶菌均具有良好的广谱抑菌能力,其对两种菌的最低抑菌质量分数分别为1.88%和2.31%。

     

  • 图  1  液料比对提取率的影响

    Figure  1.  Effects of liquid-to-solid ratio on extraction rate

    图  2  提取温度对提取率的影响

    Figure  2.  Effects of extraction temperature on extraction rate

    图  3  提取时间对提取率的影响

    Figure  3.  Effects of extraction time on extraction rate

    图  4  pH值对牛蒡叶提取率的影响

    Figure  4.  Effects of pH on extraction rate

    图  5  交互作用对提取率的影响

    a.时间与液料比Time and liquid-to-solid ratio; b.温度与液料比Temperature and liquid-to-solid ratio; c.液料比与pH Liquid-to-solid ratio and pH; d.时间与温度Time and temperature

    Figure  5.  Effects of interaction on extraction rate

    图  6  牛蒡叶提取物成分总离子流图

    Figure  6.  Total ion flow diagram of burdock leaf extract

    表  1  响应曲面实验水平设计表

    Table  1.   Level design of response surface method

    因素
    Factor
    水平Level
    -1 0 1
    A液料比Liquid-to-solid ratio/(mL·g-1) 10 20 30
    B提取温度Extraction temperature/℃ 45 50 55
    C提取时间Extraction time/min 30 60 90
    D溶剂pH值pH of extraction solution 2.0 3.0 4.0
    下载: 导出CSV

    表  2  U-HPLC流动相洗脱方式

    Table  2.   U-HPLC mobile phase elution method

    序号
    No.
    保留时间
    Retention time/min
    乙腈
    Acetonitrile/%
    0.1%甲酸溶液
    0.1% formic acid/%
    流速
    Flow velocity/(μL·min-1)
    0 0 10 90 400
    1 5 13 87 400
    2 15 20 80 400
    3 21 20 80 400
    4 30 75 25 400
    5 35 90 10 400
    6 37 100 0 400
    下载: 导出CSV

    表  3  牛蒡叶提取预实验结果

    Table  3.   Pre-test results of burdock leaf extraction

    提取方式Extraction method 提取率
    Extraction rate/%
    平均抑菌圈直径Average anti-fungal zone diameter/mm
    初提取Initial extraction 二次处理Second processing
    水提取Extraction with distilled water 30.74 8.70
    酸性水Extraction with acid water 33.08 13.17
    酸性水Extraction with acid water 萃取剩余Extraction remaining 20.38 9.91
    乙醇萃取Ethanol extraction 5.21 10.93
    下载: 导出CSV

    表  4  响应曲面因子方差分析

    Table  4.   Anova for response surface method (RSM) quadratic model analysis

    方差来源
    Variance source
    平方和
    Sum of squares
    自由度
    df
    均方
    Mean squares
    F
    F-value
    P
    P-value
    模型Model 426.13 14 30.44 22.87 < 0.000 1**
    A 23.35 1 23.35 17.54 0.000 9**
    B 3.53 1 3.53 2.656 0.126 0
    C 0.29 1 0.292 0.22 0.647 0
    D 306.62 1 306.62 230.41 < 0.000 1**
    AB 0.98 1 0.98 0.73 0.406 0
    AC 3.94 1 3.94 2.96 0.107 0
    AD 3.33 1 3.33 2.50 0.136 0
    BC 0.18 1 0.18 0.13 0.721 0
    BD 0.31 1 0.31 0.23 0.638 0
    CD 0.085 1 0.085 0.064 0.805 0
    A2 0.000 52 1 0.000 52 0.000 39 0.985 0
    B2 5.32 1 5.32 4.00 0.065 3
    C2 13.74 1 13.74 10.32 0.006 3**
    D2 58.60 1 58.60 44.04 < 0.000 1**
    残差Residual 18.63 14 1.33
    失拟项Lack of fit 17.75 10 1.77 8.02 0.029 8*
    纯误差Pure error 0.89 4 0.22
    总误差Total error 444.76 28
    注:**表示差异极显著(P < 0.01),*表示差异显著(P < 0.05)。Notes: ** means extremely significant difference(P < 0.01), * means significant difference(P < 0.05).
    下载: 导出CSV

    表  5  不同质量分数下提取物对白腐菌和褐腐菌的抑制圈直径

    Table  5.   Inhibition zone diameter of extracts against Coriolusversicolor and Gloceophyllumtrabeum under different concentrations mm

    菌种
    Wood rot fungi
    药液质量分数Medicament concentration
    对照Conrtol 2% 4% 6% 8% 10% 12% 14% 16%
    白腐菌Coriolusversicolor - - 10.72 12.73 14.48 15.40 15.67 17.64 16.06
    褐腐菌Gloceophyllumtrabeum - - 9.26 9.96 11.15 12.33 13.65 15.52 13.86
    注:-表示无明显抑菌效果。Note: - means no obvious anti-fungal effects.
    下载: 导出CSV

    表  6  牛蒡叶提取物成分分析

    Table  6.   Analysis of active components from burdock leaves

    序号
    No.
    保留时间
    Retention time/min
    母离子质荷比
    Parent ion mass-to-charge ratio
    分子式
    Molecular formula
    特征离子质荷比
    Typical ion mass-to-charge ratio
    化合物
    Compound
    1 6.31 191 C7H2O6 173、127、111 奎宁酸Quinic acid[16]
    2 12.47 153 C7H7O4 109 原儿茶酸Protocatechuic acid[17]
    3 12.80 515 C25H24O12 353、191、179 异绿原酸C Isochlorogenic acid C[18]
    4 13.64 515 C25H24O12 353、191、179 异绿原酸A Isochlorogenic acid A[18]
    5 13.89 515 C25H24O12 353、191、179 异绿原酸B Isochlorogenic acid B[18]
    6 14.55、14.84、15.20、17.66 707 353 二聚绿原酸Dimer chlorogenic acid
    7 21.02 353 C16H18O9 191、173、127 绿原酸Chlorogenic acid[16, 18]
    8 24.77 221 C15H26O 177 β-桉叶醇β-eudesmol[19]
    9 26.78 610 C27H30O16 301 芦丁Rutin[20]
    10 29.14 701 335、683
    11 29.67 449 431、269
    12 30.28 594 C27H31O15 286 5′-propanediolmatairesinoside[21]
    13 30.95 515 C25H24O12 353、191、179 4, 5-二咖啡酰奎尼酸4, 5-dicaffeoylquinic acid[18]
    14 32.18 811 793、631
    15 32.21 809 465、241
    16 33.51 327 C17H18O2S2 291、269 牛蒡酮-a Arctinone-a[12]
    下载: 导出CSV
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  • 收稿日期:  2017-01-06
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