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藏药诃子质量标志物诃子酸的分离分析及标准化研究

宋志博 陈涛 王岱杰 邹登朗 王硕 李洪梅 刘亚蓉 杨凤梅 海平 李玉林

宋志博, 陈涛, 王岱杰, 邹登朗, 王硕, 李洪梅, 刘亚蓉, 杨凤梅, 海平, 李玉林. 藏药诃子质量标志物诃子酸的分离分析及标准化研究[J]. 分析测试技术与仪器, 2021, 27(3): 165-173. doi: 10.16495/j.1006-3757.2021.03.003
引用本文: 宋志博, 陈涛, 王岱杰, 邹登朗, 王硕, 李洪梅, 刘亚蓉, 杨凤梅, 海平, 李玉林. 藏药诃子质量标志物诃子酸的分离分析及标准化研究[J]. 分析测试技术与仪器, 2021, 27(3): 165-173. doi: 10.16495/j.1006-3757.2021.03.003
SONG Zhi-bo, CHEN Tao, WANG Dai-jie, ZOU Deng-lang, WANG Shuo, LI Hong-mei, LIU Ya-rong, YANG Feng-mei, HAI Ping, LI Yu-lin. Separation and Standardization of Chebulinic Acid as Quality Marker of Tibetan Medicine Terminalia Chebula[J]. Analysis and Testing Technology and Instruments, 2021, 27(3): 165-173. doi: 10.16495/j.1006-3757.2021.03.003
Citation: SONG Zhi-bo, CHEN Tao, WANG Dai-jie, ZOU Deng-lang, WANG Shuo, LI Hong-mei, LIU Ya-rong, YANG Feng-mei, HAI Ping, LI Yu-lin. Separation and Standardization of Chebulinic Acid as Quality Marker of Tibetan Medicine Terminalia Chebula[J]. Analysis and Testing Technology and Instruments, 2021, 27(3): 165-173. doi: 10.16495/j.1006-3757.2021.03.003

藏药诃子质量标志物诃子酸的分离分析及标准化研究

doi: 10.16495/j.1006-3757.2021.03.003
基金项目: 

青海省藏药国家标准样品研制科技基础条件平台建设项目 2020-ZJ-T02

详细信息
    作者简介:

    宋志博(1995-), 男, 硕士研究生, 研究方向: 藏药物质基础和标准化研究, E-mail: 1522840147@qq.com

    通讯作者:

    海平(1964-),男,研究员,研究方向:中藏药药理及质量控制研究,E-mail:qhgxshp@163.com

    李玉林(1970-),男,博士,研究员,《分析测试技术与仪器》编委,研究方向:藏药标准化与创新药物研究,E-mail: liyulin@nwipb.cas.cn

  • 中图分类号: TQ460.7+2

Separation and Standardization of Chebulinic Acid as Quality Marker of Tibetan Medicine Terminalia Chebula

Funds: 

Construction Project of Scientific and Technological Basic Conditions Platform for the Development of Certified Reference Material of Tibetan Medicine in Qinghai Province 2020-ZJ-T02

  • 摘要: 依据GB/T 15000—2008《标准样品工作导则》的要求, 研制诃子酸国家标准样品. 以诃子的干燥成熟果实为原料, 采用大孔吸附树脂、制备型高效液相色谱技术对标准样品进行制备, 经过纯度分析、结构鉴定、均匀性检验、稳定性检验, 最后进行联合定值. 基于高效液相色谱(HPLC)技术, 采用不同检测波长进行纯度测试, 样品纯度均大于98%. 基于液相-质谱(LC-MS)进行纯度分析, 未发现明显杂质峰. 通过紫外光谱(UV)、红外光谱(IR)、质谱(MS)、核磁共振(NMR)等技术确定其结构为诃子酸. 样品均匀性良好, 4 ℃条件下24个月内稳定性良好. 定值结果确定诃子酸纯度为98.08%, 在95%置信区间范围内的相对扩展不确定度为0.50%. 研制出的诃子酸(GSB 11-3725-2020)国家标准样品可用于含量测定、检测方法评定、相关产品的检测与质量控制.
  • 图  1  样品HPLC分析色谱图

    Figure  1.  HPLC chromatogram of sample

    图  2  诃子酸标准样品制备的液相色谱分离图

    Figure  2.  HPLC separation chromatogram of standard sample of chebulinic acid

    图  3  诃子酸标准品的三维色谱图

    Figure  3.  Three-dimensional chromatograms of chebulinic acid by HPLC

    图  4  诃子酸总离子流图

    (a) 正离子模式,(b) 负离子模式

    Figure  4.  Total ionic chromatogram ofchebulinic acid by HPLC-MS

    (a) positive ion mode, (b) anion mode

    表  1  参加定值的实验室及仪器型号

    Table  1.   Laboratories and instrument models take part in certified value tests

    序号 实验室名称 HPLC仪器型号
    1 北京市理化分析测试中心 Shimadzu LC-20A
    2 中国科学院新疆理化技术研究所 Waters e2695
    3 山东大学化学与化工学院 Waters 2695
    4 山东省科学院生物研究所 Agilent 1200
    5 山东省中医药研究院 Waters e2695
    6 山东蓝城分析测试有限公司 Agilent 1200
    7 济南市出入境检验检疫局 Agilent 1260
    8 山东省分析测试中心 Agilent 1260
    下载: 导出CSV

    表  2  诃子酸的核磁共振氢谱数据

    Table  2.   1H NMR data of chebulinic acid

    基团 位置 测定值(400 MHz,MeOH-d4) 文献值(600 MHz,MeOH-d4)[25]
    Glucose 1 6.49 (1H, d, J = 2.9 Hz) 6.494 (1H, dd, J = 2.8, 0.95 Hz)
    2 5.42 (1H, brs) 5.435 (1H, ddd, J = 2.8, 1.8, 1.4 Hz)
    3 6.25 (1H, brs) 6.233 (1H, ddd, J = 3.7, 1.8, 1.0Hz)
    4 5.04 (1H, d, J = 3.1 Hz) 5.043 (1H, dd, J = 3.7, 1.1Hz)
    5 4.71 (1H, m) 4.715 (1H, dddd, J = 7.8, 6.6, 1.1, 1.0 Hz)
    6a 4.56 (1H, dd, J =10.4, 6.5 Hz) 4.639 (1H, dd, J = 11.1, 6.6Hz, )
    6b 4.77 (1H, m) 4.809(1H, dd, J = 11.1, 7.8Hz)
    Chebuloyl 2′ 4.80 (1H, d, J = 7.2 Hz) 4.817 (1H, d, J = 7.15 Hz)
    3′ 5.06 (1H, dd, J= 7.2, 1.4 Hz) 5.097 (1H, dd, J = 7.15, 1.63 Hz)
    4′ 3.87 (1H, ddd, J = 10.4, 4.0, 1.4 Hz) 3.882(1H, ddd, J=11.90, 2.97, 1.63 Hz)
    5′a 2.22 (1H, m) 2.231(1H, dd, J = 16.97, 11.90 Hz)
    5′b 2.24 (1H, m) 2.258 (1H, dd, J = 16.97, 2.97 Hz)
    2″ 7.51 (1H, s) 7.518 (1H, s)
    1-O-Galloyl-2, 6 7.11 (2H, s) 7.107 (2H, s)
    3-O-Galloyl-2, 6 7.16 (2H, s) 7.177 (2H, s)
    6-O-Galloyl-2, 6 6.98 (2H, s) 6.987 (2H, s)
    下载: 导出CSV

    表  3  诃子酸的核磁共振碳谱数据

    Table  3.   13C NMR data of chebulinic acid

    基团 位置 测定值(100 MHz,MeOH-d4) 文献值(150.9 MHz,MeOH-d4)[25]
    Glucose 1 91.6 92.94
    2 70.8 71.77
    3 61.7 62.90
    4 68.1 69.55
    5 74.9 76.14
    6 63.8 65.34
    Chebuloyl 1′ 169.4 170.80
    2′ 65.8 67.22
    3′ 40.4 41.86
    4′ 39.1 40.13
    5′ 30.1 30.83
    6′ 174.6 174.93
    7′ 173.3 174.62
    1″ 117.6 119.20
    2″ 116.2 117.77
    3″ 146.0 147.49
    4″ 139.2 140.51
    5″ 140.1 141.56
    6″ 115.0 116.26
    7″ 164.7 166.13
    1-O-Galloyl 1′′′ 118.6 120.18
    2′′′, 6′′′ 109.3 110.86
    3′′′, 5′′′ 145.2 146.63
    4′′′ 139.4 140.86
    7′′′ 164.9 166.36
    3-O-Galloyl 1′′′ 118.5 119.94
    2′′′, 6′′′ 109.3 110.92
    3′′′, 5′′′ 145.3 146.73
    4′′′ 139.4 140.94
    7′′′ 164.8 166.23
    6-O-Galloyl 1′′′ 119.3 120.87
    2′′′, 6′′′ 108.8 110.39
    3′′′, 5′′′ 145.0 146.47
    4′′′ 138.7 140.15
    7′′′ 166.5 167.96
    下载: 导出CSV

    表  4  每瓶的平均值、方差和测量次数

    Table  4.   Average value, variance and measurement times of each bottle

    瓶号 平均值/% 方差 测量次数
    1 98.24 0.000 010 3
    2 98.22 0.000 233 3
    3 98.24 0.000 300 3
    4 98.24 0.000 133 3
    5 98.23 0.000 100 3
    6 98.25 0.000 100 3
    7 98.23 0.000 033 3
    8 98.23 0.000 100 3
    9 98.24 0.000 100 3
    10 99.24 0.000 233 3
    下载: 导出CSV

    表  5  均匀性研究的方差分析

    Table  5.   Variance analysis of homogeneity test

    变差源 SS* 自由度 MS
    瓶间 0.001 453 9 0.000 161
    瓶内 0.002 867 20 0.000 143
    总和 0.005 150 29
    *:离差平方和
    下载: 导出CSV

    表  6  长期稳定性检验数据

    Table  6.   Results of long-term stability test

    时间/月 平均值/% 标准偏差/% 不确定度/%
    0 98.25 0.02 0.24
    6 98.27 0.03
    12 98.22 0.02
    18 98.22 0.02
    24 98.18 0.02
    下载: 导出CSV

    表  7  诃子酸定值结果统计分析表

    Table  7.   Statistical analysis of certified value of chebulinic acid  /%

    实验室序号 1 2 3 4 5 6 7 8
    平均值 98.19 98.28 98.17 98.20 98.22 98.23 98.26 98.13
    标准偏差 0.05 0.03 0.04 0.04 0.04 0.05 0.05 0.05
    总平均值 98.21
    Sr 0.05
    SL 0.05
    $S (\bar{\bar x} ) $ 0.02
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-07-28
  • 修回日期:  2021-09-03
  • 网络出版日期:  2021-09-29
  • 刊出日期:  2021-09-29

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