鸡骨草叶中N-反式对香豆酰酪氨酸的纯化工艺优化

Optimization of Purification Technology of N-(E)-p-coumaroyltyrosine in Leaves of Abrus cantoniensis

目的:建立鸡骨草叶中N-反式对香豆酰酪氨酸的含量测定方法,并优化其纯化工艺。方法:采用高效液相色谱法测定鸡骨草叶中N-反式对香豆酰酪氨酸的含量,色谱柱为Hypersil BDS C18,流动相为0.1%甲酸水溶液(A)-甲醇(B)(梯度洗脱),流速为1.0 mL/min,柱温为25℃,检测波长为300 nm,进样量为10μL。以聚酰胺树脂为材料,以N-反式对香豆酰酪氨酸得率为指标,采用单因素试验优化N-反式对香豆酰酪氨酸纯化的样品溶液质量浓度、上样量、静置吸附时间等条件。结果:N-反式对香豆酰酪氨酸检测进样量线性范围为2.575～51.50μg(r=0.999 9);定量限为0.000 618μg,检测限为0.000 129μg;精密度、稳定性、重复性试验的RSD均小于3%;加样回收率为97.04%～102.43%(RSD=2.06%,n=6)。最优纯化工艺为样品溶液质量浓度为0.04 g/mL(以鸡骨草叶药材计),上样量为50 m L,上样流速为1.0 m L/min,静置吸附时间为20 min,以水(含0.1%乙酸)、20%乙醇(含0.1%乙酸)、氨水(pH 10)为洗脱溶剂依次洗脱。此纯化工艺条件下,制得的N-反式对香豆酰酪氨酸的平均得率为98.94%,平均干膏含量为61.17 mg/g,平均干膏纯度为19.73%。结论:所建含量测定方法简便、准确、稳定性较好;优化所得工艺稳定、可行。

OBJECTIVE:To establish a method for content determination of N-(E)-p-coumaroyltyrosine in leaves of Abrus cantoniensis,and to optimize its purification technology.METHODS:HPLC method was adopted for the content determination of N-(E)-p-coumaroyltyrosine in A.cantoniensis.The determination was performed on Hypersil BDS C18 column with mobile phase consisted of 0.1% formic acid water(A)-methanol(B)(gradient elution)at a flow rate of 1.0 mL/min.The column temperature was set at 25℃.The detection wavelength was set at 300 nm,and sample size was 10μL.Using polyamide resin as material,the yield of N-(E)-p-coumaroyltyrosine as indicators,single factor test was used to optimize the purification technology of N-(E)-p-coumaroyltyrosine,such as concentration,sample size,stationary adsorption time.RESULTS:The linear range was 2.575-51.50μg(r=0.999 9)for N(-E)-p-coumaroyl-tyrosine.The limit of quantification was 0.000 618μg,and the detection limit was 0.000 129μg.RSDs of precision,stability and reproducibility tests were all lower than 3% .The recoveries were 97.04% -102.43% (RSD=2.06% ,n=6).The optimal purification technology was as follows:the concentration of the sample solution was 0.04 g/mL(by the leaves of A.cantoniensis);sample capacity 50 mL;the sample flow rate was 1.0 mL/min;the stationary adsorption time was 20 min;the eluting solvents were ammonia containing water(containing 0.1% acetic acid),20% ethanol(containing 0.1% acetic acid)and ammonia(pH 10).Average yield was 98.94% ,average dry paste content was 61.17 mg/g,and average dry paste purity was 19.73% by optimal purification technology.CONCLUSIONS:Established method is simple,accurate and stable.The optimized technology is stable and feasible.