蓝布正总酚酸的大孔树脂纯化工艺与质量标准研究

Optimization of purification process of total phenolic acid from Geum japonicum var.chinense by macroporous resin and investigation of its quality standard

目的:优化大孔吸附树脂富集蓝布正总酚酸的纯化工艺,建立蓝布正总酚酸部位指纹图谱,并测定二聚鞣花鞣质A、没食子酸乙酯、鞣花酸及总酚酸含量。方法:采用单因素实验,以总酚酸的吸附率、解吸附率以及总酚酸含量、转移率为指标,筛选最佳大孔树脂型号、吸附条件、除杂条件和解吸附条件;采用HPLC和紫外可见分光光度法,建立总酚酸部位指纹图谱及二聚鞣花鞣质A、没食子酸乙酯、鞣花酸和总酚酸含量测定的质量控制方法。结果:选择HPD-400型大孔树脂,最佳工艺条件为提取物上样质量浓度为8 mg·mL^(-1),吸附流速为2 BV·h^(-1),径高比为1∶8,上样量为3 BV,10%乙醇4 BV洗脱除杂,除杂流速为3 BV·h^(-1),50%乙醇5 BV洗脱,洗脱流速4 BV·h^(-1),3批次验证实验得到的总酚酸平均含量67.75%(RSD为3.02%),平均转移率为74.53%(RSD为1.20%);12批样品指纹图谱中共有个18共有峰,指认了其中6个峰,分别为二聚鞣花鞣质B、二聚鞣花鞣质G、二聚鞣花鞣质A、特里马素Ⅱ、没食子酸乙酯、鞣花酸,相似度均≥0.9,二聚鞣花鞣质A、没食子酸乙酯、鞣花酸、总酚酸含量测定方法学考察符合要求,3种成分在各自范围内线性关系良好(R2≥0.997),平均加样回收率为93.00%~103.53%,RSD为1.92%~3.87%,其12批次样品的含量范围分别为5.35%~15.25%,0.12%~0.62%,1.84%~6.27%,以没食子酸计,总酚酸线性范围良好(R2=0.995),平均加样回收率为95.30%,RSD为1.40%,其12批次样品总酚酸含量范围为53.65%~73.16%。结论:纯化工艺稳定可靠,可用于蓝布正总酚酸部位的富集。指纹图谱与含量测定方法稳定可靠,可用于蓝布正总酚酸部位的质量控制。

Objective:The purification process of total phenolic acid from Geum japonicum var.chinense using macroporous adsorption resin was optimized.The fingerprint of total phenolic acid fraction from Geum japonicum var.chinense was established accordingly.The contents of gemin A,ethyl gallate,ellagic acid and total phenolic acid were determined using the developed method. Methods: Single factor experiment was used to select the typeof macroporous resin,adsorption conditions,impurity removal conditions and desorption conditions based on theadsorption rate,resolution rate of total phenolic acid and content,transfer rate of total phenolic acid. HPLC andultraviolet-visible spectrophotometry were used to establish a quality control method for the determination of geminA,ethyl gallate,ellagic acid and total phenolic acid content,and the fingerprint analysis of total phenolic acidfraction. Results: HPD-400 macroporous resin was selected,method was: sample solution concentration of 8 mg·mL^(-1),adsorption flow rate of 2 BV·h^(-1),diameter-height ratio of 1∶ 8,sample liquid volume of 3 BV,4 BV 10% ethanolto wipe off impurity with a flow rate of 3 BV·h^(-1),5 BV 50% ethanol to elute with 4 BV·h^(-1) . The average contentof total phenolic acid obtained by the 3-batch verification test was 67. 75% ( RSD 3. 02%) ,and the average transferrate was 74. 53% ( RSD 1. 20%) . A total of 18 common peaks were established in the fingerprint,in which 6peaks were identified,namely dimerized gemin B,gemin G,gemin A,tellimagrandin II,ethyl gallate,ellagicacid,and the similarities of 12 batches of total phenolic acid fraction were above 0. 9 by comparing with the controlfingerprint. The methodology for the determination of gemin A,ethyl gallate,ellagic acid and total phenolic acidcontent met the requirements. The three components had good linear relationship in their respective ranges ( R2≥0. 997) ,and the average dosing recovery rate was 93. 00% ~ 103. 53%,RSD 1. 92% ~ 3. 87%. The contentranges of 12 batches of samples were 5. 35% ~ 15. 25%,0. 12% ~ 0. 62%,1. 84% ~ 6. 27%. In terms of gallicacid,the linear range of total phenolic acid was good ( R2 = 0. 995 ) ,the average dosing recovery rate was95. 30%,RSD was 1. 40%,and the total phenolic acid content of 12 batches of samples ranged 53. 65% ~73. 16%.Conclusion: Purification process is stable and reliable to be used for the enrichment of total phenolic acid fractionof Geum japonicum var. chinense. The fingerprints and determination method of ellagic acid content are stable andreliable to be used for the quality control of total phenolic acid fraction of Geum japonicum var. chinense.