柱前衍生HPLC测定川明参中18种氨基酸含量

Assay of 18 amino acids in Chuanmingshen violaceum roots by pre-column derivative HPLC

川明参为常用的药食两用药材,含有丰富的氨基酸。而不同产地川明参因地理位置和生态环境不同,所含氨基酸的种类和含量也不同。为此,该研究首先建立了柱前衍生HPLC测定川明参中18种氨基酸含量的方法。其次从四川省川明参的主要产地采集了24份川明参样品,根据地理位置将这些川明参样品的产地分为4个产区,测定不同产区样品中的18种氨基酸,并运用t检验及主成分分析法对这4个产区样品中的氨基酸进行综合分析。结果表明:川明参中18种氨基酸在70 min内分离良好;浓度与峰面积之间的线性关系良好(r≥0.9991,n=6);加样回收率为97.38%~101.3%(n=6),检测限为0.003~0.379μg·mL-1。说明建立的方法能准确测定川明参中多种氨基酸的含量。对川明参中氨基酸t检验的结果表明,组氨酸、胱氨酸、亮氨酸、缬氨酸、色氨酸、苯丙氨酸、苏氨酸的含量在4个产区之间具有显著性差异(P<0.05),其余氨基酸的含量差异不显著。经主成分分析法,提取前5个因子计算综合得分,4个产区得分由高至低分别为B(0.603,n=10)、C(0.2058,n=3)、A(-0.2827,n=7)、D(-1.167,n=4)产区。B区川明参中氨基酸的总量最高(12.5 mg·g-1)。由此可见,川明参含有多种氨基酸。从氨基酸总量的角度,四川阆中市、苍溪县(B区)为栽培川明参的适宜区。

The roots of Chuanmingshen violaceum is a commonly used Chinese herb and food,which contains rich amino acids.However,the kinds and amounts of amino acids are variety in this herb among the geographical location and ecological environment.Therefore,this study firstly developed a new pre-column derived HPLC method to quantify the levels of 18 amino acids in Ch.violaceum roots.Then 24 Ch.violaceum samples were harvested from its main cultivating areas in Sichuan,China.These samples were divided into 4 producing areas based on their geographical sites.The 18 kinds of amino acids were quantified in these sample by the developed method.The differences of these amino acids were further analyzed among these herbal samples and the 4 producing areas by t-test and principal component analysis(PCA).The result indicated the peaks of the 18 kinds of amino acids were separated well in 70 min.The correlation coefficients between peak areas and concentration of these amino acids were more than 0.9991(n=6).All of their recoveries were in the range of 97.38%-101.3%(n=6).Their detection limit was in the range of 0.003-0.379μg·mL-1.It demonstrates that the developed HPLC method can accurately quantify the amounts of multi-amino acids in this herb.The results of t-test analysis showed the contents of histidine,cystine,leucine,valine,tryptophan,phenylalanine and threonine were significantly different(P<0.05)among the 4 producing areas.But the differences of other amino acids were not significant.The first five factors were extracted by PCA to calculate the comprehensive score.The order of comprehensive score for the 4 producing areas was B(0.603,n=10),C(0.206,n=3),A(-0.283,n=7)and D(-1.167,n=4).The total content of amino acids in Ch.violaceum collected in B producing area was largest(12.5 mg·g-1).It is concluded the Ch.violaceum contains multi-kinds of amino acids.On the basis of amino acid amount,Langzhong city and Cangxi county in Sichuan province(producing area B)is the suitable areas for cultivating Ch.violaceum.