积雪草挥发油气相指纹图谱研究
摘要
目的:采用气相色谱法对积雪草挥发油化学成分指纹图谱进行研究,建立积雪草挥发油的气相指纹图谱标准,用于积雪草药材挥发油的质量控制,并为雪抑乐质量标准的制订提供依据。方法:采用水蒸气蒸馏法提取积雪草挥发油。利用气相色谱法测定积雪草挥发油指纹图谱,通过考察不同条件下的分离效果,优选色谱条件:色谱柱的选择,柱温或程序升温模式,载气流速,进样量;考察供试品溶剂对测定的干扰;并进一步考察指纹图谱的稳定性、精密度、重现性,最终确定积雪草指纹图谱分析的气相色谱条件。按所确定的色谱条件和测定方法,对积雪草所含挥发油成分的气相色谱指纹图谱进行研究。先后对10批积雪草药材供试品进行GC检测分析,以石竹烯为参照物,按“中药注射剂指纹图谱研究的技术要求”计算相对保留时间及峰面积比值,标定共有指纹峰,并对10批药材进行相似度比较。结果:优选出的色谱条件为:色谱柱:HP-5Ms石英毛细管柱(30m×0.25mm);柱温:初温90℃(保持5min)以10℃/min升至170℃(保持8min)以5℃/min升至250℃(保持2min);载气:高纯氮气,流量1.2ml/min(0.12Mpa);补充气:氮气,流量40ml/min;分流比:1:10;燃烧气:氢气,流量40ml/min;助燃气;空气,流量500ml/min;检测器:氢火焰离子化检测器(FID),检测器温度270℃,气化室温度235℃;进样量:1ul。供试品溶剂以选择石油醚(30～60℃)对测定无干扰。方法学考察结果:精密度试验共有峰的相对保留时间、相对峰面积的RSD为0～2.78%,重现性试验共有峰相对峰面积、相对保留时间的RSD为0.02%～2.81%,供试品在8小时之内检测稳定,共有峰的相对保留时间、相对峰面积的RSD为0.01%～2.80%。对10批积雪草药材供试品进行GC检测分析,积雪草GC指纹图谱中共有32个共有峰,共有峰峰面积总百分含量为91.62±9.12。10批药材的GC指纹图谱相似度分别为0.904～0.965。结论:本研究所建立的GC分析方法可使积雪草挥发油中各组分达到较好分离效果,精密度、重现性、稳定性良好。10批药材的GC指纹图谱相似性高,所建立的GC指纹图谱标准符合国家指纹图谱技术要求,可用于积雪草药材挥发油的质量控制,并为雪抑乐质量标准的制订提供依据。
Objective: By GC to carry out the research into the Fingerprint of the volatile oil in Centella Asiatica(L.) Urb., and to establish the standard of the volatile oil by GC Fingerprint. Method: The volatile oil in Centella asatica (L.) Urb. was extracted by wet distillation. By GC, the Fingerprint of the volatile oil in Centella asatica (L.) Urb. was determined. The separate effects under different conditions were studied so as to optimize the chromatographic condition, including the chromatographic column, the column temperature or the programmed heating mode, the flow rate of the carrier gas and the sample size. The intervention of the sample was also studied, as well as the stability, precision, reproducibility of the Fingerprint, which enabled the determination of the final chromatographic condition of the Fingerprint to Centella Asiatica(L.) Urb.. According to the optimized chromatographic condition and assay method, the Fingerprint of the volatile oil in Centella Asiatica(L.) Urb. was studied. Taking caryophyllene as the reference material, 10 batches of Centella Asiatica(L.) Urb.was assayed by GC, and the relative retention times and proportionalities of the peak area was calculated, the common peaks were marked and compared among the 10 according to the Technical Requirements for the Fingerprint Research into Chinese Crude Drug. Results: the optimal conditions were as follows: chromatographic column: HP-5Ms quartz capillary column (30mx0.25mm); column temperature: 90℃for the first 5 min, up to 170℃at the rate of 10℃/min, for 8 min, then up to 250℃at the rate of 5℃/min, for 2 min; the carrier gas: high pure nitrogen, flow rate 1.2ml/min (0.12Mpa); supplementary gas: nitrogen, flow rate 40ml/min; split ratio: 1:10; combustion air: hydrogen, flow rate 40ml/min; combustion-supporting gas: air, flow rate 500ml/min; detector: FID, detector temperature 270℃, vaporizer temperature 235℃; sample size: 1ul. Petroleum ether (30～60℃) was chosen as solvent. Results to the methodology study: the RSD of the relative retention time and the area of common peaks was 0～2.78% in the precision test, 0.02%-2.81% in the reproducibility test; 0.01%-2.80% in the stability test, which signified that the sample stayed stable within 8 hours. The Fingerprint of the 10 bathes of Centella asatica (L.) Urb. showed that there were 32 common peaks in the chromatogram, and the added area of which covered 91.62±9.12 in total percentage. Conclusion: The GC analytical method establish in this study can finely separate the ingredients in the volatile oil in Centella asatica (L.) Urb., and the precision, reproducibility and stability were satisfying. 10 batches of Centella asatica (L.) Urb. showed high similarity, and the established standard for the GC Fingerprint can meet the requirement from the guidance, thus can be applied to control the quality of Centella asatica (L.) Urb., meanwhile to provide a basis for the standard of the quality of Xue Yile.
Objective: By GC to carry out the research into the Fingerprint of the volatile oil in Centella Asiatica(L.) Urb., and to establish the standard of the volatile oil by GC Fingerprint. Method: The volatile oil in Centella asatica (L.) Urb. was extracted by wet distillation. By GC, the Fingerprint of the volatile oil in Centella asatica (L.) Urb. was determined. The separate effects under different conditions were studied so as to optimize the chromatographic condition, including the chromatographic column, the column temperature or the programmed heating mode, the flow rate of the carrier gas and the sample size. The intervention of the sample was also studied, as well as the stability, precision, reproducibility of the Fingerprint, which enabled the determination of the final chromatographic condition of the Fingerprint to Centella Asiatica(L.) Urb.. According to the optimized chromatographic condition and assay method, the Fingerprint of the volatile oil in Centella Asiatica(L.) Urb. was studied. Taking caryophyllene as the reference material, 10 batches of Centella Asiatica(L.) Urb.was assayed by GC, and the relative retention times and proportionalities of the peak area was calculated, the common peaks were marked and compared among the 10 according to the Technical Requirements for the Fingerprint Research into Chinese Crude Drug. Results: the optimal conditions were as follows: chromatographic column: HP-5Ms quartz capillary column (30mx0.25mm); column temperature: 90℃for the first 5 min, up to 170℃at the rate of 10℃/min, for 8 min, then up to 250℃at the rate of 5℃/min, for 2 min; the carrier gas: high pure nitrogen, flow rate 1.2ml/min (0.12Mpa); supplementary gas: nitrogen, flow rate 40ml/min; split ratio: 1:10; combustion air: hydrogen, flow rate 40ml/min; combustion-supporting gas: air, flow rate 500ml/min; detector: FID, detector temperature 270℃, vaporizer temperature 235℃; sample size: 1ul. Petroleum ether (30～60℃) was chosen as solvent. Results to the methodology study: the RSD of the relative retention time and the area of common peaks was 0～2.78% in the precision test, 0.02%-2.81% in the reproducibility test; 0.01%-2.80% in the stability test, which signified that the sample stayed stable within 8 hours. The Fingerprint of the 10 bathes of Centella asatica (L.) Urb. showed that there were 32 common peaks in the chromatogram, and the added area of which covered 91.62±9.12 in total percentage. Conclusion: The GC analytical method establish in this study can finely separate the ingredients in the volatile oil in Centella asatica (L.) Urb., and the precision, reproducibility and stability were satisfying. 10 batches of Centella asatica (L.) Urb. showed high similarity, and the established standard for the GC Fingerprint can meet the requirement from the guidance, thus can be applied to control the quality of Centella asatica (L.) Urb., meanwhile to provide a basis for the standard of the quality of Xue Yile.
引文
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[12]方颖等.牛至挥发油GC指纹图谱的研究[J].时珍国医国药,2007,18(7):1543-1545
[13]王磊磊等.赤芍与白芍药材高效毛细管电泳指纹图谱方法学研究[J].时珍国医国药,2008,19(10):2341-2345
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[20]王树春等.中药材熊胆X一衍射傅里叶谱分析[J].中草药,2000,31(3):214
[21]田兰等.黄芪药材的HPLC指纹图谱[J].沈阳药科大学学报,2008,25(12):979-982
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[24]游松等.银杏叶注射剂指纹图谱的研究[J].中草药,2002,33(3):216-218
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[3]徐皓.积雪草药理作用的研究[J].氨基酸和生物资源,2005,27(2):17-20
[4]赵宇新.积雪草的研究进展[J].中国中医药信息杂志,2002,9(8):81-84
[5]柴逸峰.中药指纹图谱的研究进展[J].药学服务与研究,20066(4):241-245
[6]任德权.中药指纹图谱质控技术的意义与作用.中药新药与临床药理,2001,12(3):135-140.
[7]何明芳.积雪草化学成分的研究[J].中国药科大学学报,2000,31(2):91-93
[8]陈瑶,秦路平.积雪草总苷对抑郁症大鼠神经内分泌功能的影响[J].第二军医大学学报,2002.23(11):1224-1226
[9]秦路平.积雪草挥发油成分分析及其抗抑郁作用研究[J].第二军医大学学报,1998,19(2):186-187
[10]陈瑶.积雪草提取物抗抑郁作用实验研究[J].中国药理学会通讯,2002,19(1):70
[11]Gnanapragasam A,Ebenezar KK,Sathish V,et al.Protective effect of Centella asiatica on antioxidant tissue defense system against adriamycin induced cadiomyopathy in rats[J].Life Sci,2004,76:585-586.
[12]卢茂芳.积雪草提取纯化工艺的研究[J].湖南中医药大学学报,2007,27(1):16-19
[13]史万忠.中成药中挥发油问题的探讨[J].中成药,2006,(28)11:1653-1655
[14]Z.A.AZIZ,et al.Production of asiaticoside and madecassoside in Centella asiatica in vitro and in vivo[J].Biologia Plantarum.,2007,51(1):34-42
[15]寿旦.积雪草片质量标准研究[J].浙江中医杂志,2004,39(7):310-311
[16]曾建国.HPLC法测定积雪草中三萜类化合物的含量[J].基层中药杂志,2000,14(6):7-8
[17]J.S.JANGWAN,et al.PHYTOCONSTITUENTS OF ESSENTIAL OIL OF CEISTELLA ASIATICA AND THEIR ANTIBACTERIAL ACTIVITY[J].Acta Ciencia Indica.Chemistry,2008,34(1)
[16]国家食品药品监督管理局.中药注射剂指纹图谱研究的技术要求(暂行)[S].1999.
[17]谢培山.中药色谱指纹图谱.[M].北京:人民卫生出版社,2005
[18]国家食品药品监督管理局.中药注射剂色谱指纹图谱实验研究技术指南(试行).中成药,2000,22(10):671
[19]苗爱东,孙殿甲.Excel 2002在中药指纹图谱相似度计算中的 应用[J].药学进展,2003,27(1):51-54
[1]刘小瑜等.《指纹图谱在中药研究中的应用概况》[J].天津药学,2006,18(1):46-48
[2]崔维利.《中药指纹图谱研究概况》[J].天津药学,2005,17(6):54-56
[3]李海丽.中药指纹图谱研究进展概况[J].医药卫生,2006,35(6):237-238
[4]国家药品监督管理局.中药注射剂指纹图谱研究的技术要求(暂行)[J].中成药,2000,22(10):671-675
[5]屈爱桃等.黄柏薄层色谱指纹图谱研究[J].内蒙古医学院学报, 2006,28(3):174-177
[6]李彬等.解酒肝康颗粒薄层色谱指纹图谱研究[J].世界中西医结合杂志,2007,2(10):580-581,590
[7]周漩等.高良姜薄层色谱特定(指纹)图谱的研究[J].药物分析杂志,2005,25(8):916-919
[8]钱蕙等.高效液相色谱建立掌叶大黄指纹图谱[J].天然产物研究与开发,2008,20(6):1051-1054
[9]顾中演等.灵芝药材质量的HPLC指纹图谱评价方法[J].上海大学学报:自然科学版,2008,14(6):652-655
[10]孙国祥等.HPLC指纹图谱控制复方丹参片质量[J].沈阳药科大学学报,2008,25(12):971-978
[11]王婷婷等.白芷挥发油GC指纹图谱研究[J].药物分析杂志,2007,27(9):1340-1343
[12]方颖等.牛至挥发油GC指纹图谱的研究[J].时珍国医国药,2007,18(7):1543-1545
[13]王磊磊等.赤芍与白芍药材高效毛细管电泳指纹图谱方法学研究[J].时珍国医国药,2008,19(10):2341-2345
[14]李仕海等.太子参药材的HPCE指纹谱分析[J].时珍国医国药,2007,18(4):820-821
[15]郭淘等.心舒口服液毛细管电泳指纹图谱的研究[J].中草药,2006,37(6):839-843
[16]李伟等.安徽东至中药材粗榧高效液相指纹图谱和化学成分分析[J].天然产物研究与开发,2008,20(3):461-465
[17]曹进等.桂枝汤A部分有效成分指纹图谱归属(二)[J].中国实验 方剂学杂志,2001,7(3):1-4
[18]黄从善.辛夷挥发油类成分气相色谱一质谱仪指纹图谱研究[J].湖北中医学院学报,2008,10(3):51-52
[19]王荣.甘肃道地药材大黄不同部位的指纹图谱研究[J].中国药房,2005,16(18):1435-1437
[20]王树春等.中药材熊胆X一衍射傅里叶谱分析[J].中草药,2000,31(3):214
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