血脂康抑制HMG-CoA还原酶活性的定量测定研究

Assay of potency for Xuezhikang by inhibiting HMG-CoA reductase

目的：建立血脂康的活性测定方法,用于血脂康的质量控制研究。方法：建立3-羟基-3-甲基戊二酸单酰辅酶A还原酶（HMG-Co A还原酶）酶促反应,即HMG-Co A还原酶量为3.125×10^-4U,烟酰胺腺嘌呤二核苷磷酸（NADPH）量为160 nmol·L^-1,羟甲基戊二酸单酰辅酶A（HMG-Co A）量为5 nmol·L^-1,反应时间为50 min的反应体系,加入洛伐他汀酸系列工作溶液,采用LC-MS/MS方法测定体系中产物甲羟戊酸的量,计算抑制率,得到洛伐他汀酸浓度-抑制率曲线。将血脂康样品溶液加入到酶促反应体系中,根据抑制率计算血脂康活性成分的含量。结果：洛伐他汀酸浓度为10~500 ng·m L^-1范围内抑制效应显著,质量控制样品的准确度和精密度范围分别为95.28%~101.53%,7.08%~10.51%;平均加样回收率为100.25%,RSD为2.27%。检测得到3批血脂康胶囊活性成分的含量以洛伐他汀酸计分别为每粒4.86,4.71和4.56 mg。结论：建立的方法精密度好,准确度高,可用于血脂康胶囊的生物活性测定。酶法测定结果显著高于HPLC法的测得值,推测血脂康中可能有其他成份在同时发挥抑制HMG-Co A还原酶的作用。本研究结果对血脂康的质量控制具有指导意义。

Objective ： To develop a method for potency assay of Xuezhikang, and application to quality control of Xuezhikang. Methods：HMG-CoA reductase reaction system was established, that was HMG-CoA reduetase of 3.125 ×10^-4 U,NADPH of 160 nmol·L^-1,HMG-CoA of 5 nmol·L^-1 and incubation for 50 min. LC-MS/MS method was used to determinate the HMG-CoA reduction product of mevalonic acid for calculating the inhibition rate. Using lovastatin acid as the reference,the potency was calculated from the inhibition rete of xuezhikang sam- pies to the enzyme reaction system. Results：Inhibitory effect of lovastatin acid is significant over the concentration range of 10 -500 ng· mL^-1. The accuracy and precision ranges of quality control samples were 95.28% - 101.53% and 7.08% contents of 10.51% , respectively. The average recovery for xuezhikang is 100.25% with 2.27% as RSD. The active ingredient per capsule for 3 batches of xuezhikang were 4.86,4.71 and 4.56 mg respectively which were tested using lovastatin acid as the reference. Conclusion：A precise and accurate method was established for assay of potency for xuezhikang. The results from the enzyme method are higher than that determined by HPLC method,indicating that there may be other ingredients also play the role of inhibition of HMG-CoA reductase in xuezhikang. The developed potency assay method can be applied to quality control of xuezhikang.