摘要:
OBJECTIVE: To prepare high-purity rotundic acid, and to determine the contents of syringoside, peduncloside and rotundic acid in Ilicis rotundae and its refined products. METHODS: I. rotundae saponins was prepared by ethanol extraction method and hydrolyzed with methanol and sodium hydroxide; rotundic acid monomer was obtained after recrystallization. The contents of syringoside, peduncloside and rotundic acid in I. rotundae, its refined product 1 and its refined product 2 were determined by RP-HPLC. The determination was performed on an Agilent XDB C18 column with mobile phase consisted of water-acetonitrile (gradient elution) at the flow rate of 1. 2 mL/min. The column temperature was 30 °C, and the detection wavelength was set at 210 nm. The sample size was 10 μL. RESULTS: The purity of rotundic acid exceeded 99%. The linear ranges of syringing, peduncloside and rotundic acid were 0. 18-3. 22, 0. 59-10. 36 and 0. 20-3. 53 μg, respectively (r=0. 999 9). RSDs of precision, stability (24 h) and reproducibility tests were all lower than 2. 0% (n=6-7). The average recoveries were 96. 08%-100. 81 % (RSD≤1. 98%, n=6). The contents of syringoside,peduncloside and rotundic acid in I. rotundae were 1. 61%,7. 26%, 1. 29% (RSD≤1. 08%,n=3); those of refined product 1 were 0,82. 59%,4. 18% (RSD≤1. 67%,n=3); those of refined product 2 were 0, 73. 29%, 7. 41% (RSD≤1. 15%,n=3),respectively. CONCLUSIONS: Prepared rotundic acid has high purity and is simple in preparation, safe and environmentally-friendly. RP-HPLC method that established is simple and reliable, and can meet the requirements for rapid analysis of I. rotundae and its refined products.%目的:制备高纯度的救必应酸并测定救必应药材及精制品中3种活性成分紫丁香苷、长梗冬青苷、救必应酸的含量.方法:采用醇提法制备救必应皂苷,以甲醇和氢氧化钠进一步水解,并经重结晶后得到救必应酸单体.采用反相高效液相色谱(RP-HPLC)法测定救必应药材及精制品1和精制品2中紫丁香苷、长梗冬青苷、救必应酸的含量.色谱柱为Agilent XDB C18,流动相为水-乙腈(梯度洗脱),柱温为30 °C,流速为1. 2 mL/min,检测波长为210 nm,进样量为10 μL.结果:所制救必应酸纯度达99%以上.紫丁香苷、长梗冬青苷、救必应酸进样量分别在0. 18~3. 22、0. 59~10. 36、0. 20~3. 53 μg范围内与各自峰面积积分值呈良好线性关系(r均为0. 9999),精密度、稳定性(24 h)、重复性试验的RSD均小于2. 0% (n=6~7),平均加样回收率为96. 08%~100. 81% (RSD≤1. 98%,n=6).紫丁香苷、长梗冬青苷、救必应酸的含量在救必应药材中分别为1. 61%、7. 26%、1. 29% (RSD≤1. 08%, n=3), 在精制品1中分别为0、82. 59%、4. 18% (RSD≤1. 67%, n=3);在精制品2中分别为0、73. 29%、7. 41% (RSD≤1. 15%, n=3).结论:所制救必应酸纯度高、制备方法简单且安全环保.建立的RP-HPLC法简单、可靠,可满足快速测定救必应药材及其精制品含量的要求.