Application Research Of QAMS In Hawthorn Leaf And Its Preparations

Hawthorn leaf is the dry leaf of Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge.var.major N. E. Br. which belongs to rosaceae family. The name of Hawthorn was appeared in Compendium of Materia Medica firstly. The Hawthorn was recorded as “red claw” in Tang Materia Medica before Compendium of Materia Medica. The Hawthorn leaf extract was first collected in Chinese pharmacopoeia of 2005 edition. The major constituents of Hawthorn leaf are flavonoids, organic acids and some trace elements and so on. They have been shown to treat irregular heartbeat, chest pain, congestive heart failure, hardening of the arteries and high blood pressure. Their preparations have been applied more and more widely in clinical such as Yixintong tablet, Shanmei capsule and Xin’an capsule, etc. So Hawthorn leaf is an important traditional Chinese medicine(TCM) that have great value in clinical application.At present, the quality control of TCM usually uses the model of determining one or multiple constituents. But the model has been limited in practical production and market supervision because of the reference substance supplied shortages and high cost. Aiming at this problem, QAMS which is a method of determining multi-components by one reference substance was appeared firstly from the team of Zhimin Wang. The rhizoma coptidis of 2010 Chinese pharmacopoeia also use this method for quality control. So this study intents to establish QAMS for determining the six constituents of chlorogenic acid(a), Vitexin-glucoside(b), Vitexin-rhamnoside(c), Vitexin(d), Rutin(e), Hyperoside(f) at the same time. And then QAMS was applied to Hawthorn leaf extract and preparations such as Yixintong tablet and Xin’an capsule, so as to realize the all round quality control in Hawthorn leaf, Hawthorn leaf extract and their preparations. It can provide a new quality control model for other TCM. Part one QAMS method was established in Hawthorn leafObjective:To establish the quantitative analysis of multi-components by single-marker(QAMS) method for the simultaneous determination of chlorogenic acid(a), Vitexin-glucoside(b), Vitexin-rhamnoside(c), Vitexin(d), Rutin(e), Hyperoside(f) in hawthorn leaf. The external standard method(ESM) was used to determine the content at the same time, in order to verify the accuracy and feasibility of QAMS methodMethods:1. Using the Agilent 1200 High performance liquid chromatography(HPLC), the internal standard Vitexin-glucoside(b) and the following chromatographic conditions to establish QAMS method in Hawthorn leaf: Agilent ZORBAX SB-C18 column(4.6 mm×250 mm, 5 μm), 0.1% formic acid(A)-acetonitrile(B)-tetrahydrofuran(C) were mobile phases to do the gradient elution: 0~20~30~40~50 min, A(90%~84%~84%~78%~74%), B(8%~ 14%~14%~20%~24%), C(2%~2%~2%~2%~2%); The detection wavelength was set at 350 nm. The flow rate was 1.0 m L·min-1. The column temperature was maintained at 30 ℃. The injection volume of the samples was 10 μL.2. The contents determined by QAMS method and traditional ESM were analyzed by relative error.Results:1. The values of RCF were 1.119, 1.009, 0.706, 1.063, 0.830 with Vitexin-glucoside as the internal standard.2. Using the ratio of the relative retention time to locate the chromatographic peak of other five constituents, RSD<5%.3. The RSD of RCF were less than 5% with different instruments, chromatographic column or in different laboratories; The RSD of RCF were less than 5% with temperature and flow velocity changing in small scope; The RSD of chlorogenic acid and Vitexin-rhamnoside were between 5% and 10% and the RSD of other three constituents were less than 5% when the detection wavelength changes in small scope.4. The relative errors of the results from QAMS method were less than 5% compared with ESM.Conclusion:It was successful to establish QAMS method in hawthorn leaf that can realize the determination of six constituents content with only one reference substance. There was no significant differences in content results compared QAMS method to ESM and it was shown that QAMS method was accurate, reliable and strong feasibility. Part two Application of QAMS method in Hawthorn leaf extractObjective:QAMS method which has been established successfully in hawthorn leaf was applied to determine the content of six constituents in hawthorn leaf extract and so as to control the quality from the multi-target constituents.Methods:The Chromatographic conditions and testing method were the same as “Part one”. The results were validated by ESM at the same time.Results:The relative errors of QAMS method were less than 5% compared with ESM.Conclusion:There was no significant difference in content results of hawthorn leaf extract compared with QAMS method and ESM. Part three Application of QAMS method in hawthorn leaf preparationsObjective:QAMS method which has been established successfully in hawthorn leaf was applied to determine the content of six constituents in preparations made by hawthorn leaves or their extract and so as to improve their quality standard of Yixintong tablets and Xin’an capsules.Methods:The Chromatographic conditions and testing method were the same as “Part one”. The results were validated by ESM at the same time.Results:1. The results compared with QAMS method and ESM in Yixintong tablets are as follows: The relative errors of Vitexin were more 5% in five batches of Yixintong tablets from Shanxi Hengshan Traditional Chinese Medicine Co.,LTD; The relative errors of chlorogenic acid were more than 5% in nine batches of Yixintong tablets from Shanxi cooperates Taisheng Pharmaceutical Co.,LTD, Hainan Tianhuang Pharmaceutical Co.,LTD and Shanxi Angsheng Pharmaceutical Co.,LTD, respectively. The relative errors of others were less than 5%.2. The results compared with QAMS method and ESM in Xin’an capsules are as follows: the relative error of Vitexin was bigger than 5% in batches of 130501, 131003 from Changchun Xinyu Pharmaceutical Co.,LTD; the relative error of Hyperoside was more than 10% in three batches from Jilin Wantong Pharmaceutical Co.,LTD. The relative errors of others were less than 5%.Conclusion:There were differences in content results of chlorogenic acid, Vitexin, Hyperoside that were from some samples compared with QAMS method and ESM.