Analysis of marker compounds with anti-platelet aggregation effects in Mailuoning injection using platelet binding assay combined with HPLC-DAD-ESI-MS and solid-phase extraction technique.
BACKGROUND
Mailuoning is prepared from a traditional formula of Chinese medicines and widely used as an antithrombotic agent. In this study, the platelet binding assay was used as a novel biospecific separation and analysis method to explore its active constituents, which could be considered as marker compounds for quality control.


OBJECTIVE
To establish a rapid and simple method to predict marker compounds in herbal medicine injection and evaluate the effects of those compounds.


METHODS
Platelets were used to bind and separate constituents. Binding constituents were analysed and taken as potential active compounds for further evaluation. Solid-phase-extraction was adopted to improve sensitivity. HPLC-DAD and ESI-MS were used to determine the binding constituents.


RESULTS
Five compounds were extracted through the platelet binding process and identified as neochlorogenic acid, caffeic acid, isochlorogenic acid and their isomers. Caffeic acid was selected for the flow cytometric assay to test its effect on platelets activation, which was determined by CD62P (P-selectin) expression. The results indicated that caffeic acid could significantly inhibit platelet activation while chlorogenic acid did not.


CONCLUSIONS
Caffeic acid could be considered as a marker compound of Mailuoning injection due to its anti-platelet effect. The study also suggested that platelet binding assay combined with some preconcentration technique could be efficiently used to predict anti-platelet compounds in complicated herbal medicines.
Yu
Li
L
Jiangsu Key laboratory for TCM formulae Research, Nanjing University of Chinese Medicine, Nanjing, China.
Li
Yingbin
Y
Fan
Hongwei
H
Duan
Jinao
J
Zhu
Quan
Q
Li
Shaoping
S
D000925:Anticoagulants / Q000737:chemistry* / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004365:Drugs, Chinese Herbal / Q000737:chemistry*; D062385:Hydroxybenzoates; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000925:Anticoagulants; D004365:Drugs, Chinese Herbal; D062385:Hydroxybenzoates; C017616:phenolic acid