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Standardization of Chebulagic Acid as Quality Marker of Tibetan Medicine Terminalia Chebula
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摘要: 以藏药诃子为原料，研制诃黎勒酸国家标准样品. 诃子经60%乙醇提取，大孔树脂富集，制备液相色谱技术得到诃黎勒酸标准品. 采用高效液相色谱（HPLC）、液相色谱-质谱（LC-MS）和薄层色谱（TLC）等手段进行纯度分析. 采用紫外光谱（UV）、红外光谱（IR）、高分辨质谱（MS）和核磁共振波谱（NMR）技术进行结构确认. 最后进行均匀性、稳定性和联合定值. 结果表明，诃黎勒酸样品均匀性良好，4 ℃储存，24个月内稳定. 联合定值确定诃黎勒酸纯度标准值为99.33%，符合国家标准品的要求. 研制出的诃黎勒酸（GSB 11-3724-2020）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.Abstract: A certified reference material of chebulagic acid has been developed using a Tibetan medicine Terminalia Chebula as the raw material. Terminalia Chebula was extracted with 60% ethanol, enriched by the macroporous resin and through the preparative liquid chromatographic purification to obtain chebulagic acid. The purity analysis was carried out by high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and thin-layer chromatography (TLC). The structure was identified using ultraviolet (UV) spectroscopy, infrared (IR) spectroscopy, mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy. Finally, the homogeneity, stability and cooperative certification were carried out. The results indicated that the chebulagic acid has a good uniformity and a fine stability within 24 months at 4 ℃. The standard value of the purity of chebulagic acid was 99.33%, which was in accordance with the requirements of the certified reference material. The developed chebulagic acid (GSB 11-3724-2020) can be used for the content determination, method evaluation, testing and quality control of related products.
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Key words:
- chebulagic acid /
- reference material /
- homogeneity /
- stability /
- certification
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图 3 诃黎勒酸标准样品制备图
Figure 3. Preparative chromatogram of standard sample of chebulagic acid
图 4 诃黎勒酸薄层色谱图
（a）展开剂为乙酸乙酯∶甲醇∶三氟乙酸（体积比5∶1∶0.01），（b）展开剂为丙酮∶甲醇∶三氟乙酸（体积比4∶1.25∶0.01）
Figure 4. Thin layer chromatograms of chebulagic acid
图 6 诃黎勒酸总离子流图
（a）正离子模式，（b）负离子模式
Figure 6. Totals ionic diagrams of chebulagic acid
(a) positive ion mode，（b）anion mode
图 7 诃黎勒酸质谱图
（a）正离子模式，（b）负离子模式
Figure 7. MS spectra of chebulagic acid
(a) positive ion mode, (b) anion mode
表 1 仪器型号
Table 1. Models of instruments
仪器名称 仪器型号 公司 液相色谱质谱仪 Agilent 6520 Q-TOF 美国安捷伦公司 高效液相色谱仪 Agilent 1260 美国安捷伦公司 大孔树脂纯化柱 50 L 浙江温兄机械阀业有限公司 循环超声提取仪 CTXNW-100B 北京弘祥隆生物技术股份有限公司 红外光谱仪 Vertex 70 德国布鲁克公司 核磁共振波谱仪 BRUKER 400 MHz 德国布鲁克公司 热重分析仪 SHIMADZU TG-40 DTA-40M 日本岛津公司 高效制备液相色谱仪 LC-8A岛津 日本岛津公司 冷冻干燥机 GLZ-1B 上海浦东冷冻干燥设备有限公司 
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表 2 参加定值实验室及仪器型号
Table 2. Laboratories participated in calibration and models of instrument
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
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表 3 诃黎勒酸的核磁共振氢谱信号
Table 3. 1H NMR data of chebulagic acid
化合物 位置 测定值(400 MHz，MeOH-d4) 文献值(500 MHz，MeOH-d4)[21] Glucose 1 6.50 (1H, s) 6.47 (1H, s) 2 5.39 (1H, brs) 5.46 (1H, s) 3 5.82 (1H, brs) 5.86 (1H, s) 4 5.23 (1H, d, J= 3.0 Hz) 5.19 (1H, d, J=3.6 Hz) 5 4.81 (1H, overlap) 4.80 (1H, t, J=17.4 Hz) 6a 4.37 (1H, dd, J=10.2, 7.8 Hz) 4.38 (1H, dd, J=10.8, 8.0 Hz,) 6b 4.86 (1H, overlap) 4.70 (1H, t, 20.3 Hz) Chebuloyl 2' 4.79 (1H, d, J=7.4 Hz) 4.84 (1H, d, J=7.2 Hz) 3' 5.04 (1H, d, J=7.4, 1.2 Hz) 5.04 (1H, dd, J=7.3, 1.5 Hz) 4' 3.80 (1H, m) 3.80 (1H, m) 5' 2.15(2H, m) 2.11 (2H, d, J=4.5 Hz) 2'' 7.48 (1H, s) 7.46 (1H, s) 1-O-Galloyl-2,6 7.07 (2H, s) 7.11 (2H, s) HHDP (3-O end) 6.84 (2H, s) 6.98 (2H, s) HHDP (6-O end) 6.63 (2H, s) 6.62 (2H, s)
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表 4 诃黎勒酸的碳谱数据
Table 4. 13C NMR data of chebulagic acid
化合物 位置 测定值(100 MHz，MeOH-d4) 文献值(150.9 MHz，MeOH-d4)[21] 化合物 位置 测定值(100 MHz，MeOH-d4) 文献值(150.9 MHz，MeOH-d4)[21] Glucose 1 91.1 91.7 1-O-Galloyl 1''' 118.7 119.7 2 69.7 70.7 2''', 6''' 109.5 110.4 3 61.0 61.8 3''', 5''' 145.1 145.9 4 65.6 66.3 4''' 139.5 140.0 5 72.9 73.7 7''' 164.9 165.3 6 63.3 63.9 HHDP
(3-O end)1 114.6 115.2 Chebuloyl 1' 169.4 169.8 2 124.2 125.2 2' 65.4 66.2 3 106.8 107.5 3' 40.3 40.9 4 144.8 145.3 4' 38.6 39.2 5 136.2 136.2 5' 29.2 29.9 6 143.9 144.6 6' 173.6 173.6 7 168.7 168.7 7' 173.0 173.2 HHDP
(6-O end)1' 116.3 117.1 1'' 114.8 115.7 2' 123.1 124.2 2'' 117.6 118.4 3' 109.1 110.1 3'' 116.2 116.9 4' 144.2 145.1 4'' 146.0 146.5 5' 137.3 137.6 5'' 139.0 139.7 6' 144.1 144.8 6'' 140.0 140.8 7' 166.1 166.4 7'' 165.0 165.6
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表 5 均匀性检验的测量数据
Table 5. Measurement data of homogeneity test
/% 瓶号 1 2 3 4 5 6 7 8 9 10 第1份 99.49 99.49 99.50 99.48 99.46 99.50 99.48 99.49 99.49 99.48 第2份 99.49 99.46 99.51 99.48 99.49 99.51 99.49 99.47 99.48 99.50 第3份 99.47 99.46 99.47 99.50 99.47 99.49 99.48 99.48 99.49 99.49 平均值 99.48 99.47 99.49 99.49 99.47 99.50 99.48 99.48 99.49 99.49 总体平均值 99.49
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表 6 均匀性研究的方差分析表
Table 6. Variance analysis of homogeneity test
变差源 方差和（SS） 自由度 均方差（MS） 瓶间 0.002 1 9 0.000 24 瓶内 0.003 2 20 0.000 16 总和 0.005 3 29
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表 7 短期稳定性检验试验结果
Table 7. Data of short-term stability test
温度/℃ 平均值/% 标准偏差/% 不确定度/% −20 99.37 0.02 0.04 2~8 99.42 0.02 20 99.44 0.02 40 99.39 0.02 60 99.34 0.02
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表 8 长期稳定性检验数据
Table 8. Data of long-term stability test
时间/月 平均值/% 标准偏差/% 不确定度/% 0 99.45 0.02 0.24 6 99.39 0.03 12 99.42 0.03 18 99.35 0.02 24 99.38 0.02
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表 9 诃黎勒酸定值结果统计分析表
Table 9. Statistical analysis of certified value of chebulagic acid
/% 实验室序号 1 2 3 4 5 6 7 8 平均值 99.60 99.58 99.57 99.52 99.45 99.47 99.44 99.39 标准偏差 0.09 0.04 0.06 0.04 0.06 0.02 0.04 0.05 x 99.50 Sr 0.05 SL 0.08 $S\left(\overline{\overline{x} }\right)$ 0.03
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表 10 不确定度
Table 10. Table of uncertainty
/% 样品名称 不确定度 定值 均匀性 短期稳定性 长期稳定性 水分 灰分 合成 扩展 诃黎勒酸 0.03 0.02 0.04 0.24 0.02 0.02 0.25 0.50
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