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化风丹药母指纹图谱的建立及不同发酵时间样品中7种核苷类成分的含量测定
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| 篇名： | 化风丹药母指纹图谱的建立及不同发酵时间样品中7种核苷类成分的含量测定 |
| TITLE： | Establishment of HPLC Fingerprint of Huafengdan Yaomu and Content Determination of 7 Nucleosides in Samples of Different Fermentation Time |
| 摘要： | 目的：建立化风丹药母的指纹图谱，并测定不同发酵时间样品中7种核苷类成分的含量。方法：采用高效液相色谱法（HPLC），色谱柱为PntulipsBP-C18，流动相为甲醇-水（梯度洗脱），流速为0.8mL/min，检测波长为260nm，柱温为35℃，进样量为10μL。以黄嘌呤为参照，绘制12批化风丹药母的HPLC指纹图谱；采用《中药色谱指纹图谱相似度评价系统（2012版）》进行相似度评价，确定共有峰；同法测定不同发酵时间（0、1、2、3、4周）样品中尿嘧啶、次黄嘌呤、黄嘌呤、尿苷、肌苷、鸟苷、胸苷的含量。结果：12批化风丹药母共有8个共有峰，相似度为0.712～0.954；指认了7种成分，分别为尿嘧啶、次黄嘌呤、黄嘌呤、尿苷、肌苷、鸟苷、胸苷。不同发酵时间样品中上述7种成分检测质量浓度的线性范围分别为0.87～8.7μg/mL（r＝0.9996）、1.51～15.1μg/mL（r＝0.9997）、6.08～60.8μg/mL（r＝0.9995）、1.52～15.2μg/mL（r＝0.9996）、1.82～18.2μg/mL（r＝0.9996）、1.48～14.8μg/mL（r＝0.9996）、1.63～16.3μg/mL（r＝0.9993）；定量限分别为0.0274、0.0763、0.2504、0.1723、0.1011、0.0783、0.0842μg/mL，检测限分别为0.0087、0.0255、0.0079、0.0841、0.0357、0.0269、0.0275μg/mL；精密度、重复性、稳定性（12h）试验的RSD均小于3％；加样回收率分别为94.16％～100.16％（RSD＝2.24％，n＝6），93.87％～100.65％（RSD＝2.67％，n＝6），93.52％～99.66％（RSD＝2.30％，n＝6），93.67％～98.24％（RSD＝1.89％，n＝6），96.00％～102.18％（RSD＝1.96％，n＝6），94.62％～101.54％（RSD＝2.82％，n＝6），97.72％～104.56％（RSD＝2.97％，n＝6）。发酵0～4周时，上述7种成分及总核苷的含量分别为0.042～0.232、0.027～0.181、0.039～0.651、0.026～0.225、0.034～0.111、0.009～0.124、0.079～0.099、0.647～1.292mg/g；发酵1～4周时的尿嘧啶、次黄嘌呤、黄嘌呤及总核苷的含量均较发酵0周时显著升高，而尿苷、肌苷、鸟苷的含量较发酵0周时显著降低。结论：所建指纹图谱特征性强且操作简便，可用于化风丹药母的质量控制；含量测定方法准确、可靠，可用于同时测定其中7种核苷类有效成分的含量；化风丹药母中的核苷类成分含量受发酵时间的影响。 |
| ABSTRACT： | OBJECTIVE：To establish fingerprint of Huafengdan yaomu ，and to determine the contents of 7 nucleosides in samples of different fermentation time. METHODS ：HPLC method was adopted. The determination was performed on Pntulips BP-C18 column with mobile phase consisted of methanol-water （gradient elution ）at the flow rate of 0.8 mL/min. The detection wavelength was set at 260 nm，and column temperature was 35 ℃. The sample size was 10 μL. Using xanthine as reference， HPLC fingerprint of 12 batches of Huafengdan yaomu was drawn. The similarity of samples were evaluated with Similarity Evaluation System of TCM Chromatographic Fingerprints （2012 edition）. Common peaks were confirmed. The contents of uracil ， hypoxanthine，xanthine，uridine，inosine，guanosine and thymidine were determined in samples of different fermentation time （0， 1，2，3，4 weeks）by the same method. RESULTS ：There were 8 common peaks in 12 batches of Huafengdan yaomu ，with similarities ranging from 0.712 to 0.954；7 components were identified ，namely uracil ，hypoxanthine，xanthine，uridine，inosine， guanosine and thymidine. The linear ranges of mass concentrations of above 7 components in samples at different fermentation time were 0.87-8.7 μ g/mL （r＝0.999 6）， 4030 1.51-15.1 μg/mL（r＝0.999 7），6.08-60.8 μg/mL（r＝0.999 5）， 号） 1.52-15.2 μg/mL（r＝0.999 6），1.82-18.2 μg/mL（r＝0.999 6）， 1.48-14.8 μg/mL（r＝0.999 6），1.63-16.3 μg/mL（r＝0.999 3）. The limits of quantification were 0.027 4，0.076 3，0.250 4，0.172 3，0.101 1，0.078 3，and 0.084 2 μ g/mL，and the detection limits were 0.008 7，0.025 5，0.007 9，0.084 1，0.035 7，0.026 9，0.027 5 μg/mL，respectively. RSDs of precision ， repeatability and stability tests （12 h）were all less than 3％. The sample recovery rates were 94.16％-100.16％（RSD＝2.24％，n＝ 6），93.87％-100.65％（RSD＝2.67％，n＝6），93.52％-99.66％（RSD＝2.30％，n＝6），93.67％-98.24％（RSD＝1.89％，n＝6）， 96.00％-102.18％（RSD＝1.96％，n＝6），94.62％-101.54％（RSD＝2.82％，n＝6），97.72％-104.56％（RSD＝2.97％，n＝6）. After fermentation for 0-4 weeks，the contents of the above 7 components and total nucleosides were 0.042-0.232，0.027-0.181， 0.039-0.651，0.026-0.225，0.034-0.111，0.009-0.124，0.079-0.099，0.647-1.292 mg/g，respectively. After fermentation for 1-4 weeks，the contents of uracil ，hypoxanthine，xanthine and total nucleosides were significantly increased ，compared with 0 week of fermentation；the contents of uridine ，inosine and guanosine were significantly lower than those in 0 weeks. CONCLUSIONS ：The established fingerprint has strong characteristics and simple to operate ，which can be used for the quality control of Huafengdan yaomu；the content determination method is accurate and reliable ，and can be used to simultaneously determine the contents of 7 active nucleosides ；the content of nucleosides in Huafengdan muyao is affected by fermentation time. |
| 期刊： | 2021年第32卷第02期 |
| 作者： | 曹国琼，游珊丽，徐剑，张永萍 |
| AUTHORS： | CAO Guoqiong ，YOU Shanli，XU Jian，ZHANG Yongping |
| 关键字： | 化风丹药母；高效液相色谱法；指纹图谱；核苷类成分；含量测定 |
| KEYWORDS： | Huafengdan yaomu ；HPLC；Fingerprint；Nucleosides；Content determination |
| 阅读数： | 252 次 |
| 本月下载数： | 15 次 |
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