目的:对羊胆中的胆汁酸类成分进行考察,并对其中的主要胆汁酸进行测定,最后对研究结果进行深入讨论。方法:首先对收集到的来源确切的羊胆样品进行冷冻干燥处理,以确保干燥粉末与新鲜胆汁成分的一致性。然后使用TLC法,分别采用异辛烷-乙醚-冰醋酸-正丁醇-水(10∶5∶5∶3∶0.8)和异辛烷-乙醚-冰醋酸-正丁醇-水(6∶5∶5∶3∶1)为展开剂,使用10%硫酸乙醇溶液显色,明确羊胆中的胆汁酸成分,根据斑点的深浅对胆汁酸含量进行初步的判断;再使用Agilent SB-C18色谱柱(250 mm×4.6 mm, 5 μm),10 mmol·L-1乙酸铵水溶液-乙腈为流动相进行梯度洗脱,蒸发光散射检测器检测,对羊胆中2个含量最高的胆汁酸类成分牛磺胆酸和牛磺去氧胆酸进行了含量测定研究。结果:本研究明确了羊胆中含有胆酸、甘氨胆酸、甘氨去氧胆酸、甘氨鹅去氧胆酸、牛磺胆酸、牛磺去氧胆酸、牛磺鹅去氧胆酸和牛磺石胆酸,其中以牛磺胆酸和牛磺去氧胆酸含量为最高。测得10批羊胆样品的牛磺胆酸和牛磺去氧胆酸的平均含量分别为47.5%和8.8%,这2个成分的总含量平均为56.3%,牛磺胆酸含量与牛磺去氧胆酸含量的平均比值为5.4。结论:本研究所建立的TLC方法不仅可用于羊胆的研究,同样适合用作其他胆汁类药材的鉴别或胆汁酸类成分的确定研究,而定量的HPLC-ELSD法也可以简便、快速、准确且经济地用作羊胆的含量测定工作。
Objective: To investigate bile acids in sheep bile, quantify the major bile acids in it, and discuss these research results. Methods: Firstly, the samples of fresh sheep bile were processed with freeze drying to samples of powder to ensure consistency of ingredients. In the study on the categories of bile acids, TLC methods with isooctane-diethyl ether-acetic acid-n-butanol-water(10∶5∶5∶3∶0.8) and isooctane-diethyl ether-acetic acid-n-butanol-water(6∶5∶5∶3∶1) as developing solvent and 10% sulfuric acid ethanol solution as chromogenic agent were performed. Two major bile acids, including taurocholic acid (TCA) and taurodeoxycholic acid (TDCA), in sheep bile were quantified by HPLC-ELSD using Agilent SB-C18 column(250 mm×4.6 mm, 5 μm) with 10 mmol·L-1 ammonium acetate aqueous solution-acetonitrile as gradient elution mobile phase. Results: In the study, the categories of bile acids were made clear. There were cholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, TCA, TDCA, taurochenodeoxycholic acid and taurolithocholic acid in sheep bile. The average contents of TCA, TDCA and the sum contents of the two bile acids were 47.5%, 8.8% and 56.3%, respectively. The average ratio of TCA and TDCA was 5.4. Conclusion: The TLC method in the study can be used for the study on identification of other animal bile medicinal materials and the categories of bile acids determination. The HPLC-ELSD method can be used for the quantification of TCA and TDCA in sheep bile due to its simplicity, speed and accuracy.
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